U.S. patent application number 12/934735 was filed with the patent office on 2011-11-24 for treatment of metabolic-related disorders using hypothalamic gene transfer of bdnf and compositions therefor.
This patent application is currently assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION. Invention is credited to Lei Cao, Matthew J. During.
Application Number | 20110288160 12/934735 |
Document ID | / |
Family ID | 41114778 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110288160 |
Kind Code |
A1 |
During; Matthew J. ; et
al. |
November 24, 2011 |
Treatment of Metabolic-Related Disorders Using Hypothalamic Gene
Transfer of BDNF and Compositions Therefor
Abstract
Described herein is a system which uses a gene therapy particle
that includes at least one gene, cDNA, fragment or analogue of at
least one neurotropin that binds to the trkB receptor or the trkB
receptor itself. The gene therapy particle is capable of being
delivered to a subject in need thereof for therapy of a metabolic
disorder. In a particular aspect, this invention is directed to an
inventive method that demonstrates a remarkable efficacy on
lowering body weight.
Inventors: |
During; Matthew J.;
(Columbus, OH) ; Cao; Lei; (Columbus, OH) |
Assignee: |
THE OHIO STATE UNIVERSITY RESEARCH
FOUNDATION
Columbus
OH
|
Family ID: |
41114778 |
Appl. No.: |
12/934735 |
Filed: |
March 27, 2009 |
PCT Filed: |
March 27, 2009 |
PCT NO: |
PCT/US2009/038593 |
371 Date: |
February 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61072146 |
Mar 27, 2008 |
|
|
|
Current U.S.
Class: |
514/44R ;
435/320.1; 435/455 |
Current CPC
Class: |
A61P 3/10 20180101; C12N
15/1136 20130101; C12N 2310/14 20130101; C12N 2830/50 20130101;
C12N 15/86 20130101; C12N 2750/14143 20130101; A61P 3/04 20180101;
C12N 2330/51 20130101; A61P 3/00 20180101; A61P 3/08 20180101; A61K
48/0075 20130101; A61K 48/005 20130101; C12N 2830/48 20130101 |
Class at
Publication: |
514/44.R ;
435/320.1; 435/455 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/85 20060101 C12N015/85; C12N 15/86 20060101
C12N015/86; A61P 3/08 20060101 A61P003/08; A61P 3/00 20060101
A61P003/00; A61P 3/10 20060101 A61P003/10; A61P 3/04 20060101
A61P003/04; C12N 15/63 20060101 C12N015/63; C12N 15/861 20060101
C12N015/861 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support from the
National Institutes of Health research grant NS44576 and the
government has certain rights in the invention.
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. A gene therapy particle comprising a vector expression
cassette, a regulatory gene sequence, and a nucleotide sequence
encoding a brain derived neurotrophic factor (BDNF), derivative or
functional fragment thereof wherein the nucleotide sequence
comprises one or more of: SEQ ID NO:1, SEQ ID NO:2, the DNA
sequence represented by AGRP484 (484 bp, -133 bp to +351 bp from
the start of the noncoding exon28) in SEQ ID NO:7, and the DNA
sequence represented by AGRP814 (814 bp, -463/+351) in SEQ ID NO:7,
or a derivative or functional fragment thereof; an amino acid
sequence at least 90% homologous thereto; or an amino acid sequence
at least 85% homologous thereto.
11. (canceled)
12. The gene therapy particle of claim 10, wherein the nucleotide
sequence encodes TrkB or a variant capable of transducing BDNF
effects.
13. The gene therapy particle of claim 10, wherein the vector
comprises an adeno-associated viral vector, lentiviral vector or
adenoviral vector.
14. The gene therapy particle of claim 10, wherein the vector is an
adeno-associated viral vector selected from the serotype of one or
more of: AAV-1, AAV-2, AAV-3, AAV-4, AAAV-5, AAV-6, AAV-7, AAV-8,
AAV-9 and AAV-10.
15. The gene therapy particle of claim 14, wherein the vector is
any human or non-human primate isolate, variant, recombinant,
chimeric or AAV capsid, including mutations, substitutions,
deletions or additions.
16. The gene therapy particle of claim 14, wherein the
adeno-associated viral vector is AAV-2, or a modified form of AAV-2
with an altered tropism.
17. The gene therapy particle of claim 14, wherein the AAV
nucleotide sequences are derived from AAV serotype 1 (AAV-1).
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. The gene therapy particle of claim 10, wherein the nucleotide
sequence is a human BDNF protein sequence.
23. A pharmaceutical composition comprising the gene therapy
particle of claim 10, in a biocompatible pharmaceutical
carrier.
24. A method of gene therapy for the treatment of a subject having
a mutation or polymorphism in the BDNF gene comprising:
administering a therapeutically effective amount of a recombinant
gene therapy particle to cells of the subject, wherein the gene
therapy particle comprises the gene particle of claim 10.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. The method of claim 24, wherein the gene therapy particle is
administered by a stereotactic route.
30. The method of claim 24, wherein the gene therapy particle is
administered by an approach via the subject's brain ventricles,
with or without an endoscope.
31. The method of claim 30, wherein the gene therapy particle is
administered by via the subject's lateral ventricle, through to the
third ventricle which lies immediately adjacent to the
hypothalamus.
32. The method of claim 24, wherein the gene therapy particle is
administered by a transnasal approach.
33. The method of claim 24, wherein the gene therapy particle is
administered transphenoidally, in which a direct approach through
the subject's nasal sinuses to the base of the brain, and then a
device inserted into the nasal sinuses to deliver the vector
directly through this skull base approach into the
hypothalamus.
34. The method of claim 24, wherein the gene therapy particle is
administered into the subject's ventricles with sufficient
hypothalamic expression obtained to induce weight loss.
35. The method of claim 24, wherein the cells of the subject are
brain hypothalamic cells.
36. The method of claim 24, wherein the cells of the subject are
hypothalamic cells responsible for the control of food intake
and/or the subject's metabolism.
37. The method of claim 24, wherein the subject is a primate.
38. The method of claim 24, wherein the subject is a human.
39. The method of claim 24, wherein the particle is in a
biocompatible pharmaceutical carrier.
40. A method of reducing or eliminating metabolic-related disorder
symptoms comprising administering to a subject in need thereof a
therapeutically effective amount of the gene therapy particle of
claim 10.
41. The method of claim 40, wherein the metabolic-related disorder
symptoms are one or more of obesity, insulin sensitivity, syndrome
X and diabetes.
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. A method for delivering a nucleotide sequence to a mammalian
nervous system target cell, the method comprising the step of:
administering an expression vector to the target cell, wherein the
expression vector comprises a nucleotide sequence encoding brain
derived neurotrophic factor (BDNF), or a derivative or functional
fragment thereof, wherein the nucleotide sequence comprises one or
more of: SEQ ID NO:1, SEQ ID NO:2, the DNA sequence represented by
AGRP484 (484 bp, -133 bp to +351 bp from the start of the noncoding
exon28) in SEQ ID NO:7, and the DNA sequence represented by AGRP814
(814 bp, -463/+351) in SEQ ID NO:7, or a derivative or functional
fragment thereof; an amino acid sequence at least 90% homologous
thereto; or an amino acid sequence at least 85% homologous
thereto.
53. (canceled)
54. (canceled)
55. (canceled)
56. The method of claim 52, wherein expression of BDNF, or a
derivative or functional fragment thereof, in the target cell
reduces symptoms associated with a metabolic disorder.
57. The method of claim 56, wherein the metabolic disorder is one
or more of obesity, insulin sensitivity, syndrome X and
diabetes.
58. The method of claim 52, wherein the expression vector is a
viral or a non-viral expression vector.
59. The method of claim 58, wherein the viral expression vector is
an adeno-associated virus (AAV) vector.
60. The method of claim 58, wherein the viral expression vector is
an AAV vector capable of transducing the target cell and the AAV
vector is free of both wildtype and helper virus.
61. The method of claim 60, wherein the AAV vector is a serotype 2
AAV vector or a chimeric serotype 1/2 AAV vector.
62. (canceled)
63. (canceled)
64. (canceled)
65. (canceled)
66. The method of claim 52, wherein the nucleotide sequence
encoding BDNF, or a derivative or functional fragment thereof, is
operably linked to an inducible regulatory sequence.
67. The method of claim 66, wherein the inducible regulatory
sequence renders BDNF expression central nervous
system-specific.
68. The method of claim 52, wherein the target cell is a mammalian
cell.
69. The method of claim 52, wherein the target cell is a human
cell.
70. The method of claim 52, wherein the target cell is in cell
culture.
71. The method of claim 52, wherein the target cell is in a living
mammal.
72. (canceled)
73. (canceled)
74. (canceled)
75. The method of claim 52, wherein the administering is by
stereotaxic microinjection.
76. An AAV vector which retains only the replication and packaging
signals of AAV, and which comprises a nucleotide sequence encoding
BDNF, or a derivative or a functional fragment thereof, wherein the
nucleic acid sequence comprises a nucleic acid sequence of SEQ ID
NO:1, SEQ ID NO:2, AGRP484 (484 bp, -133 bp to +351 bp from the
start of the noncoding exon28) in SEQ ID NO:7 or AGRP814 (814 bp,
-463/+351) in SEQ ID NO:7, or a derivative or a functional fragment
thereof.
77. (canceled)
78. A composition comprising an AAV vector of claim 76 and a
pharmaceutically acceptable carrier.
79. A method for treating a mammal with a metabolic disorder, the
method comprising the step of: administering an expression vector
to a target cell in the mammal, wherein the expression vector
comprises a nucleic acid sequence encoding BDNF, or a derivative or
functional fragment thereof, wherein the nucleic acid sequence
encoding BDNF is a nucleic acid sequence encoding an amino acid
sequence comprising SEQ ID NO:1, SEQ ID NO:2, AGRP484 (484 bp, -133
bp to +351 bp from the start of the noncoding exon28) in SEQ ID
NO:7 or AGRP814 (814 bp, -463/+351) in SEQ ID NO:7 or a derivative
or a functional fragment thereof; or an amino acid sequence at
least 90% homologous thereto; or an amino acid sequence at least
85% homologous thereto, and wherein the administering results in
expression of BDNF, or a derivative or functional fragment thereof,
in the target cell and the expression reduces the symptoms of the
metabolic disorder, thereby treating the mammal with such
disorder.
80. The method of claim 79, wherein the expression vector is a
viral or a non-viral expression vector.
81. The method of claim 79, wherein the viral expression vector is
an adeno-associated virus (AAV) vector.
82. (canceled)
83. The method of claim 79, wherein the metabolic disorder is
obesity.
84. The method of claim 79, wherein the administering is by
stereotaxic microinjection.
85. (canceled)
86. (canceled)
87. (canceled)
88. (canceled)
89. (canceled)
90. (canceled)
91. (canceled)
92. (canceled)
93. (canceled)
94. A rAAV vector comprising a BDNF transgene flanked by two loxP
sites (flox-BDNF), capable of being subsequently knocked out by a
second viral vector delivering Cre recombinase.
95. The rAAV vector of claim 94, encoding a GFP-Cre fusion
protein.
96. A method for ablating loxP-modified genes in the brain,
including the hypothalamus, with low toxicity, comprising
administering an effective amount of the rAAV vector of claim
95.
97. The gene therapy particle of claim 10, wherein the vector
expression cassette includes a promoter selected from: chicken
.beta.-actin (CBA), agouti related protein 484 (AGRP484), and
agouti related protein 84 (AGRP814).
98. (canceled)
99. (canceled)
100. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/072,146, filed Mar. 27, 2008, the disclosure of
which is expressly incorporated herein by reference.
TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION
[0003] Described herein is a system which uses a combination of a
transgene with a physiological regulated microRNA to that same
transgene. In one particular aspect, the system uses one vector to
deliver both the transgene and an interfering RNA targeting the
same transgene. In another particular aspect, this invention is
directed to an inventive method that demonstrates a remarkable
efficacy on lowering body weight.
BACKGROUND OF THE INVENTION
[0004] Obesity and the related condition, syndrome X (or metabolic
syndrome) represent two of the most significant causes of morbidity
in the western world, and their incidences are growing rapidly.
Lifestyle and non-pharmacological approaches have limited efficacy,
and particularly in the megaobese (30% or greater of ideal body
weight, IBW), pharmacological treatments are being used including
stimulants, appetite suppressants and drugs that interfere with fat
absorption. Moreover, bariatric surgery has increased dramatically
reflecting just how problematic and refractory severe obesity is to
alternative less invasive strategies. Bariatric surgery is often
effective but comes with significant morbidity in its own right,
hence there is a need for a safer and more effective approach.
[0005] Gene expression studies have been conducted in the
hypothalamus of animals which lived in an optimized enriched
environment (where such animals have increased physical and social
activity which results in a much healthier and resilient animal).
These animals have improved insulin sensitivity, have reduced fat
mass and, despite ad libitum food, do not gain weight to obese
levels as do control standard housed animals. The inventors'
previous gene expression studies showed a consistent elevation in
BDNF in the hypothalamus at 2, 4 and 9 weeks of enrichment. Many
other genes changed at various timepoints of enrichment but were
not consistently upregulated.
[0006] There is a need, therefore, for a method to regulate the
metabolic and related disorders without the drawbacks associated
with the currently recommended treatments.
[0007] Considering the above-mentioned, there is also a need for
therapeutic strategies to treat such metabolic and related
disorders.
SUMMARY OF THE INVENTION
[0008] The invention is based, at least in part, on the inventors'
discovery that gene transfer of brain-derived neurotrophic factor
(BDNF) to the hypothalamus can lead to an insulin-sensitive, lean
and healthy phenotype in ad libitum fed animals.
[0009] In a first aspect, there is provided herein a gene therapy
particle comprising: one or more of: i) a nucleotide sequence
encoding at least one neurotropin, or a derivative or functional
fragment thereof, that binds to a receptor capable of being
delivered to a subject in need thereof for therapy of a metabolic
disorder; ii) a nucleotide sequence encoding at least one receptor
for the neurotropin, or a derivative or functional fragment
thereof, that binds to a receptor capable of being delivered to a
subject in need thereof for therapy of a metabolic disorder; and
iii) a nucleotide sequence that mediates or facilitates the
signaling of at least one neurotropin, or a derivative or
functional fragment thereof, that binds to a receptor capable of
being delivered to a subject in need thereof for therapy of a
metabolic disorder.
[0010] In certain embodiments, the neurotropin comprises a brain
derived neurotrophic factor (BDNF), or a derivative or functional
fragment thereof.
[0011] In certain embodiments, the receptor comprises a receptor
(trkB) or any analogue or variant capable of transducing one or
more of the neurotropin's effects.
[0012] In certain embodiments, the gene therapy particle targets a
nucleotide sequence encoding at least one neurotropin.
[0013] In certain embodiments, the gene therapy particle targets
any flanking 5' or 3' sequence of the nucleotide sequence encoding
at least one neurotropin, including untranslated sequences.
[0014] In certain embodiments, the receptor comprises a trkB
receptor and the neurotropin comprises BDNF.
[0015] In another aspect, there is provided herein a gene therapy
particle comprising a combination of a transgene with a
physiological regulated RNA to that same transgene, or with a
transgene mRNA including all untranslated 3' and 5' sequences, the
gene therapy particle being capable of being delivered to a subject
in need thereof for therapy of a metabolic disorder. In certain
embodiments, the interfering RNA can comprise one or more of: a
micro RNA, a short hairpin (shRNA) or short interfering (siRNA),
and any other form of interfering RNA, including but limited to a
WPRE sequence.
[0016] In another aspect, there is provided herein a gene therapy
particle comprising a vector expression cassette, a regulatory gene
sequence, and a nucleotide sequence encoding a brain derived
neurotrophic factor (BDNF), derivative or functional fragment
thereof.
[0017] In another aspect, there is provided herein a recombinant
gene therapy particle comprising a nucleotide sequence containing a
vector expression cassette having an enhancer and promoter, a
regulatory gene sequence, wherein the nucleotide sequence encodes a
brain derived neurotrophic factor (BDNF), derivative or functional
fragment thereof, and wherein the nucleotide sequence is inserted
to one or more cloning sites between the promoter and the
regulatory sequence.
[0018] In certain embodiments, the nucleotide sequence encodes TrkB
or any variant capable of transducing BDNF effects.
[0019] In certain embodiments, the vector is an adeno-associated
viral vector, lentiviral vector or adenoviral vector. In certain
embodiments, the vector is an adeno-associated viral vector
selected from the serotype of one or more of: AAV-1, AAV-2, AAV-3,
AAV-4, AAAV-5, AAV-6, AAV-7, AAV-8, AAV-9 and AAV-10. In certain
embodiments, the vector is any human or non-human primate isolate,
variant, recombinant, chimeric or AAV capsid, including mutations,
substitutions, deletions or additions. In certain embodiments, the
adeno-associated viral vector is AAV-2, or a modified form of AAV-2
with an altered tropism. In certain embodiments, the AAV nucleotide
sequences are derived from AAV serotype 1 (AAV-1).
[0020] In certain embodiments, the nucleotide sequence includes the
DNA sequence represented by SEQ ID NO:1.
[0021] In certain embodiments, the nucleotide sequence includes the
DNA sequence represented by AGRP484 (484 bp, -133 bp to +351 bp
from the start of the noncoding exon28) in SEQ ID NO:7.
[0022] In certain embodiments, the nucleotide sequence includes the
DNA sequence represented by AGRP814 (814 bp, -463/+351) in SEQ ID
NO:7.
[0023] In certain embodiments, the nucleotide sequence is a human
BDNF protein sequence.
[0024] In another aspect, there is provided herein a pharmaceutical
composition comprising the gene therapy particle, in a
biocompatible pharmaceutical carrier.
[0025] In another aspect, there is provided herein a method of gene
therapy for the treatment of a subject having a mutation or
polymorphism in the BDNF gene comprising: administering a
therapeutically effective amount of a recombinant gene therapy
particle to cells of the subject, wherein the gene therapy particle
comprises the gene particle.
[0026] In certain embodiments, the gene therapy particle comprises
the sequence represented by SEQ ID NO:1. In certain embodiments,
the gene therapy particle comprises the sequence represented by
AGRP484 (484 bp, -133 bp to +351 bp from the start of the noncoding
exon28) in SEQ ID NO:7. In certain embodiments, the gene therapy
particle comprises the sequence represented by AGRP814 (814 bp,
-463/+351) in SEQ ID NO:7.
[0027] In certain embodiments, the gene therapy particle is
administered by a stereotactic route. In certain embodiments, the
gene therapy particle is administered by an approach via the
subject's ventricles, with or without an endoscope. In certain
embodiments, the gene therapy particle is administered via the
subject's lateral ventricle, through to the third ventricle which
lies immediately adjacent to the hypothalamus. In certain
embodiments, the gene therapy particle is administered via a
transnasal approach. In certain embodiments, the gene therapy
particle is administered transphenoidally, in which a direct
approach through the nasal sinuses to the base of the brain, and
then a device inserted to deliver the vector directly through this
skull base approach into the hypothalamus. In certain embodiments,
the gene therapy particle is administered into the subject's
ventricles with sufficient hypothalamic expression obtained to
induce weight loss.
[0028] In certain embodiments, the cells of the subject are brain
hypothalamic cells. In certain embodiments, the cells of the
subject are hypothalamic cells responsible for the control of food
intake and/or the subject's metabolism. In certain embodiments, the
subject is a primate. In certain embodiments, the subject is a
human.
[0029] In certain embodiments, the particle is in a biocompatible
pharmaceutical carrier.
[0030] In another aspect, there is provided herein a method of
reducing or eliminating metabolic-related disorder symptoms
comprising administering to a subject in need thereof a
therapeutically effective amount of the gene therapy particle. In
certain embodiments, the metabolic-related disorder symptoms are
one or more of obesity, insulin sensitivity, syndrome X and
diabetes.
[0031] In another aspect, there is provided herein a method for
ameliorating a symptom of a metabolic system disorder in a mammal,
the method comprising direct administration of an adeno-associated
virus-derived vector to a target cell in the brain of the mammal,
the vector comprising a DNA sequence, wherein the DNA sequence is
exogenous to an adeno-associated virus and comprises a sequence
encoding a therapeutic protein in operable linkage with a promoter
sequence, wherein the adeno-associated virus-derived vector is free
of both wild-type and helper virus, and wherein the exogenous DNA
sequence is expressed in the target cell such that the symptom of
the metabolic disorder is ameliorated.
[0032] In certain embodiments, the exogenous DNA sequence is
expressed in the target cell either constitutively or under
regulatable conditions.
[0033] In certain embodiments, the exogenous DNA sequence encodes a
brain derived neurotrophic factor (BDNF) protein.
[0034] In certain embodiments, all adeno-associated viral genes of
the vector have been deleted or inactivated.
[0035] In certain embodiments, the vector comprises only the
inverted terminal repeats of adeno-associated virus.
[0036] In certain embodiments, the promoter sequence is a central
nervous system-specific promoter. In certain embodiments, the
target cell is a primate target cell. In certain embodiments, the
primate target cell is a human target cell.
[0037] In certain embodiments, direct administration is by
stereotaxic injection.
[0038] In another aspect, there is provided herein an
adeno-associated virus-derived vector comprising only the
replication and packaging signals of adeno-associated virus, and
further comprising a nucleotide sequence encoding a brain derived
neurotrophic factor (BDNF), or a derivative or functional fragment
thereof, and a promoter sequence.
[0039] In another aspect, there is provided herein a method for
delivering a nucleotide sequence to a mammalian nervous system
target cell, the method comprising administering an expression
vector to the target cell, wherein the expression vector comprises
a nucleotide sequence encoding brain derived neurotrophic factor
(BDNF), or a derivative or functional fragment thereof.
[0040] In certain embodiments, the nucleotide sequence encoding
BDNF, or a derivative or functional fragment thereof, is expressed
in the target cell either constitutively or under regulatable
conditions.
[0041] In certain embodiments, expression of BDNF, or a derivative
or functional fragment thereof, in the target cell alters neuronal
excitability.
[0042] In certain embodiments, expression of BDNF, or a derivative
or functional fragment thereof, in the target cell reduces neuronal
excitability.
[0043] In certain embodiments, expression of BDNF, or a derivative
or functional fragment thereof, in the target cell reduces symptoms
associated with a metabolic disorder.
[0044] In certain embodiments, the metabolic disorder is one or
more of obesity, insulin sensitivity, syndrome X and diabetes.
[0045] In certain embodiments, the expression vector is a viral or
a non-viral expression vector. In certain embodiments, the viral
expression vector is an adeno-associated virus (AAV) vector. In
certain embodiments, the viral expression vector is an AAV vector
capable of transducing the target cell and the AAV vector is free
of both wildtype and helper virus.
[0046] In certain embodiments, the AAV vector is a serotype 2 AAV
vector or a chimeric serotype 1/2 AAV vector.
[0047] In certain embodiments, the nucleotide sequence comprises
SEQ ID NO:1.
[0048] In certain embodiments, the nucleotide sequence comprises
AGRP484 (484 bp, -133 bp to +351 bp from the start of the noncoding
exon28) in SEQ ID NO:7.
[0049] In certain embodiments, the nucleotide sequence comprises
AGRP814 (814 bp, -463/+351) in SEQ ID NO:7.
[0050] In certain embodiments, the nucleotide sequence encoding
BDNF, or a derivative or functional fragment thereof, is operably
linked to an inducible regulatory sequence.
[0051] In certain embodiments, the inducible regulatory sequence
renders BDNF expression central nervous system-specific. In certain
embodiments, the target cell is a mammalian cell. In certain
embodiments, the target cell is a human cell. In certain
embodiments, the target cell is in cell culture. In certain
embodiments, the target cell is in a living mammal.
[0052] In certain embodiments, the method includes delivering
nucleic acid encoding BDNF to cells of the nervous system to effect
expression of BDNF in cells of the nervous system to treat a
metabolic disorder. In certain embodiments, the metabolic-related
disorder symptoms are one or more of obesity, insulin sensitivity,
syndrome X and diabetes.
[0053] In certain embodiments, the nucleic acid sequence encoding
BDNF is a nucleic acid sequence encoding an amino acid sequence
comprising SEQ ID NO:1, AGRP484 (484 bp, -133 bp to +351 bp from
the start of the noncoding exon28) in SEQ ID NO:7 or AGRP814 (814
bp, -463/+351) in SEQ ID NO:7, or a derivative or functional
fragment thereof; an amino acid sequence at least 90% homologous
thereto; or an amino acid sequence at least 85% homologous thereto.
In certain embodiments, the administering is by stereotaxic
microinjection.
[0054] In another aspect, there is provided herein an AAV vector
which retains only the replication and packaging signals of AAV,
and which comprises a nucleotide sequence encoding BDNF, or a
derivative or a functional fragment thereof. In certain
embodiments, the AAV vector, wherein the nucleic acid sequence
comprises a nucleic acid sequence of SEQ ID NO:1, AGRP484 (484 bp,
-133 bp to +351 bp from the start of the noncoding exon28) in SEQ
ID NO:7 or AGRP814 (814 bp, -463/+351) in SEQ ID NO:7, or a
derivative or a functional fragment thereof.
[0055] In another aspect, there is provided herein a composition
comprising an AAV vector and a pharmaceutically acceptable
carrier.
[0056] In another aspect, there is provided herein a method for
treating a mammal with a metabolic disorder, the method comprising
administering an expression vector to a target cell in the mammal,
wherein the expression vector comprises a nucleic acid sequence
encoding BDNF, or a derivative or functional fragment thereof, and
wherein the administering results in expression of BDNF, or a
derivative or functional fragment thereof, in the target cell and
the expression reduces the symptoms of the metabolic disorder,
thereby treating the mammal with such disorder. In certain
embodiments, the expression vector is a viral or a non-viral
expression vector. In certain embodiments, the viral expression
vector is an adeno-associated virus (AAV) vector. In certain
embodiments, the nucleic acid sequence encoding BDNF is a nucleic
acid sequence encoding an amino acid sequence comprising SEQ ID
NO:1, AGRP484 (484 bp, -133 bp to +351 bp from the start of the
noncoding exon28) in SEQ ID NO:7 or AGRP814 (814 bp, -463/+351) in
SEQ ID NO:7, or a derivative or a functional fragment thereof; or
an amino acid sequence at least 90% homologous thereto; or an amino
acid sequence at least 85% homologous thereto. In certain
embodiments, the metabolic disorder is obesity. In certain
embodiments, the administering is by stereotaxic
microinjection.
[0057] In another aspect, there is provided herein a method for
delivering a nucleotide sequence to a mammalian nervous system
target cell, the method comprising administering an
adeno-associated virus (AAV) vector to the target cell, wherein the
vector transduces the target cell; and wherein the AAV vector
comprises an AAV vector, and is free of both wildtype and helper
virus.
[0058] In another aspect, there is provided herein a method for
treating a mammal with a metabolic disorder, the method comprising
administering an adeno-associated virus (AAV) vector to a target
cell in the mammal, wherein the AAV vector comprises an AAV vector,
and wherein the administering results in expression of BDNF, or a
derivative or functional fragment thereof, in the target cell and
the expression reduces the symptoms of the metabolic disorder,
thereby treating the mammal with such disorder.
[0059] In certain embodiments, the method includes a single dosing
to which the mammal responds, and yet adapts and autoregulates
regardless of the mammal's diet.
[0060] In certain embodiments, the metabolic disorder is obesity,
and the reduction of symptoms includes one or more of: loss of
liver steatosis, improvement in insulin sensitivity, improvement in
glucose tolerance, and reversal of hyperleptinemia and lipid
dyslipidemia.
[0061] In another aspect, there is provided herein a method for
regulation of a given functional transgene of interest, wherein the
transgene is self-regulated by a microRNA driven by one or more
promoters activated, in turn, by a physiological change induced by
the transgene of interest.
[0062] In another aspect, there is provided herein a method for
regulating one or more genes encoding proteins involved in energy
expenditure in a subject in need thereof, comprising upregulating
Ucps in the subject's liver.
[0063] In another aspect, there is provided herein a method for
causing reversible weight gain, comprising knocking out a BDNF
transgene by expression of Cre.
[0064] In another aspect, there is provided herein an
autoregulatory negative feedback system comprising using RNAi
coupled with a transgene for inducing one or more physiological
changes.
[0065] In another aspect, there is provided herein a knockout
system comprising using delivery of a second, rescue vector,
comprising using a loxP-Cre recombination system to knock out a
nucleotide sequence encoding for BDNF or a derivative or functional
fragment thereof.
[0066] In another aspect, there is provided herein a rAAV vector
comprising a BDNF transgene flanked by two loxP sites (flox-BDNF),
capable of being subsequently knocked out by a second viral vector
delivering Cre recombinase.
[0067] In certain embodiments, the rAAV vector encodes a GFP-Cre
fusion protein.
[0068] In another aspect, there is provided herein a method for
ablating loxP-modified genes in the brain, including the
hypothalamus, with low toxicity, comprising administering an
effective amount of the rAAV vector.
[0069] In certain embodiments, the vector expression cassette
includes a promoter selected from: chicken .beta.-actin (CBA),
agouti related protein 484 (AGRP484), and agouti related protein 84
(AGRP814).
[0070] In another aspect, there is provided herein an rAAV plasmid
comprising a vector expression cassette consisting of an enhancer,
a promoter, a regulatory element and bovine growth hormone
polyadenosine flanked by AAV inverted terminal repeats, wherein
fused human BDNF cDNA is fused at the 5' terminus and then inserted
into at least one multiple cloning site between the promoter and
the sequence.
[0071] In another aspect, there is provided herein an rAAV plasmid
comprising a vector expression cassette consisting of a
cytomegalovirus enhancer, a chicken .beta.-actin (CBA) promoter, a
woodchuck post-transcriptional regulatory element (WPRE) and bovine
growth hormone polyadenosine flanked by AAV inverted terminal
repeats, wherein fused human BDNF cDNA is fused at the 5' terminus
and then inserted into multiple cloning sites between the CBA
promoter and the WPRE sequence.
[0072] In certain embodiments, the rAAV plasmid includes a weaker
promoter to drive the BDNF, wherein, in the obese state the AGRP
promoter is dialed right down, but is activated when weight is
lost, and wherein, at target weight, the AGRP promoter is stronger
than the promoter driving the BDNF.
[0073] Various objects and advantages of this invention will become
apparent to those skilled in the art from the following detailed
description of the preferred embodiment, when read in light of the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIGS. 1a-1f: FIG. 1: Hypothalamic gene delivery of BDNF
leads to weight loss, changes in serum biomarkers and hypothalamic
gene expression in wild-type mice fed with NCD.
[0075] FIG. 1a: EGFP fluorescence. Scale bar, 200 .mu.m. ARC,
arcuate nucleus; VMH, ventromedial hypothalamus; DMH, dorsalmedial
hypothalamus; 3V, third ventricle.
[0076] FIG. 1b: Immunoreactivity to HA tag. Scale bar, 200
.mu.m.
[0077] FIG. 1c: Colocalization of HA (red) with NPY (green) in
arcuate nucleus. Scale bar, 10 .mu.m.
[0078] FIG. 1d: Body weight in GFP-expressing (n=10) and
BDNF-expressing mice (n=14), *P<0.0001.
[0079] FIG. 1e: Perigonadal white adipose tissue weight (n=8
GFP-expressing mice and n=10 BDNF-expressing mice,
*P<0.001).
[0080] FIG. 1f: Gene expression in hypothalamus (n=4 per group, P
values are shown on the bars). Ntrk2, neurotrophic tyrosine kinase,
receptor, type 2; Sgk1, serum/glucocorticoid-regulated kinase-1;
Vgf, nerve growth factor inducible.
[0081] FIGS. 2a-2h: Hypothalamic gene delivery of BDNF prevents DIO
in wild-type mice.
[0082] FIG. 2a: Body weight (n=9 GFP-expressing mice and n=10
BDNF-expressing mice, *P<0.0001). Diet was switched from NCD to
HFD on day 10 after rAAV injection.
[0083] FIG. 2b: Two months HFD feeding led to abdominal obesity in
YFP-expressing mice but not in BDNF-expressing mice. Arrow shows
pericardial fat absent in the BDNF-expressing mouse. Scale bar, 1
cm.
[0084] FIG. 2c: Fat pad weight 72 d after rAAV injection in mice
fed on HFD for 2 months (n=8 per group, *P<0.0001).
[0085] FIG. 2d: H&E-stained WAT section showing the smaller
size of adipose cells in BDNF-expressing mice compared to
YFP-expressing mice. Scale bar, 300 .mu.m.
[0086] FIGS. 2e-2f: Hepatic steatosis was prevented by BDNF
treatment, as shown by oil red O (FIG. 2e) and H&E (FIG. 2f)
staining. Scale bar, 300 .mu.m.
[0087] FIGS. 2g-2h: Glucose tolerance test after overnight fast
(n=4 per group, P<0.0001 for both glucose concentration (FIG.
2g) and insulin (FIG. 2h) concentration).
[0088] FIGS. 3a-3c: Gene expression profiles of HFD-fed mice.
Relative mRNA expression levels of the indicated genes in WAT (FIG.
3a), liver (FIG. 3b) and hypothalamus (FIG. 3c (n=6 per group).
Bars show the relative expression in BDNF-expressing mice as
compared to YFP-expressing mice. P values are shown over the bars.
Srebf1, sterol regulatory element-binding transcription factor-1;
Rb1, retinoblastoma-1; Dio2, deiodinase, iodothyronine, type
II.
[0089] FIGS. 4a-4h: Autoregulatory BDNF vector to treat db/db
mice.
[0090] FIG. 4a: Schematic of the rAAV vectors. The autoregulatory
vector contains two expression cassettes, one to express BDNF under
a constitutive promoter, the other to express a microRNA targeting
the same transgene driven by a promoter (AGRP484) responsive to
BDNF-induced physiological changes. PolyA, polyadenosine
sequence.
[0091] FIG. 4b: Body weight of db/db mice (n=8 YFP-expressing mice,
n=7 BDNF-miR-scr-expressing mice and n=9 BDNF-miR-Bdnf-expressing
mice; P<0.0001 for comparisons between each pair of groups).
[0092] FIG. 4c: Mice receiving BDNF-miR-Bdnf remained lean 3 months
after rAAV injection. Scale bar, 1 cm.
[0093] FIG. 4d: Food intake in BDNF-treated mice compared to
YFP-expressing mice. P values are shown over the bars.
[0094] FIG. 4e: Liver weight and fat pad weight in YFP-expressing
and BDNF-miR-Bdnf-expressing mice (n=8 each group,
*P<0.001).
[0095] FIGS. 4f-4g: Glucose tolerance test on mice after overnight
fast (n=6 YFP-expressing mice and n=5 BDNF-miR-Bdnf-expressing
mice; P<0.05 for glucose concentration (FIG. 4f) and P<0.0001
for insulin concentration (FIG. 4g).
[0096] FIG. 4h: Biomarkers in serum 1 month after rAAV injection
(n=8 YFP-expressing mice, n=4 BDNF-miR-scr-expressing mice and n=9
BDNF-miR-Bdnf-expressing mice; *P<0.001, +P<0.05 and
#P=0.06). IGF-1, insulin-like growth factor-1.
[0097] FIGS. 5a-5h: BDNF-induced weight loss is reversible by
Cre-loxP-mediated knockout of the transgene.
[0098] FIG. 5a: Weight change of wild-type mice after first rAAV
injection (n=10 YFP-expressing mice and n=24 flox-BDNF-expressing
mice, P<0.01).
[0099] FIGS. 5b-5d: Effect of BDNF treatment on energy expenditure.
Energy expenditure (heat, FIG. 5b), physical activity (FIG. 5c) and
respiratory exchange ratio (RER, FIG. 5d) were significantly
increased in BDNF-expressing mice (n=5 YFP-expressing mice and n=6
flox-BDNF-expressing mice, P<0.05) during both light and dark
cycles.
[0100] FIG. 5e: Glucose tolerance test on mice after overnight fast
(n=7 YFP-expressing mice and n=9 flox-BDNF-expressing mice, 3 weeks
after first rAAV injection; P<0.05).
[0101] FIG. 5f: Weight change of mice since second rAAV injection
(n=10 mice given YFP and Cre, n=7 mice given flox-BDNF and empty
vector and n=14 mice given flox-BDNF and Cre; P<0.05
flox-BDNF+Cre versus flox-BDNF+empty).
[0102] FIG. 5g: Body mass index (BMI) of mice 4 months after the
first surgery (n=5 mice given YFP and Cre, n=4 mice given flox-BDNF
and empty vector and n=6 mice given flox-BDNF and Cre; P values are
shown above the bars).
[0103] FIG. 5h: Volumetric bone mineral density (vBMD) of whole
body (excluding skull) and right femur, as measured by
microcomputed tomography scan (n=3 mice given YFP and n=4 mice
given flox-BDNF).
[0104] FIGS. 6a-6c: Food intake was not changed by hypothalamic
gene transfer of BDNF in mice fed on standard diet (FIG. 6a) or
high fat diet (FIG. 6b).
[0105] FIG. 6c: Hypothalamic gene transfer of BDNF improved insulin
tolerance test in diet induced obesity model. Insulin was injected
to mice without a fast and blood glucose concentration was measured
(n=4 YFP, n=5 BDNF, P<0.0001).
[0106] FIGS. 7a-7e: rAAV mediated RDNF overexpression did not cause
cytotoxicity. rAAV-BDNF was injected unilaterally to hypothalamus
with no cell loss as shown in Nissl staining (FIG. 7a), no gliosis
as shown in GFAP staining (FIG. 7b) compared to counterlateral
side. TUNEL assay showed no apoptosis in hypothalamus injected with
rAAV-BDNF or rAAV-Cre.
[0107] FIG. 7c: TUNEL assay positive control counterstained with
DAPI (FIG. 7d) unilateral injection of rAAV-BDNF. (FIG. 7e)
bilateral injection of rAAV-Cre. Scale bars 200 .mu.m.
[0108] FIG. 8: MicroRNA vector knocked down hypothalamic BDNF
expression and led to accelerated weight gain. In vitro experiments
showed that the microRNA vector knocked down BDNF mRNA by 65% and
protein levels by 80%. We further assessed the efficacy of this
microRNA to BDNF by generating a rAAV vector with miR-Bdnf driven
by CBA promoter. We also generated a control microRNA vector
targeting a scrambled sequence (miR-scr) against no known genes. We
injected rAAV vectors of miR-Bdnf or miR-scr bilaterally into the
hypothalamus of wild-type mice and fed the mice on standard diet.
Quantitative RT-PCR and ELISA showed that the miR-Bdnf vector
significantly reduced BDNF expression in hypothalamus at both mRNA
(a, *P<0.01) and protein levels compared to miR-scr (b,
*P<0.01). This reduction of BDNF expression in hypothalamus led
to accelerated weight gain in miR-Bdnf mice by 26 days after
injection (c, *P<0.01). n=10-23 per group.
[0109] FIG. 9a: Hypothalamic gene expression profile of db/db mice
treated with an autoregulatory BDNF vector. Relative mRNA
expression levels of the indicated genes in hypothalamus are shown
as percentage of control mice (n=6 YFP, n=9 BDNF-miR-Bdnf).
[0110] FIG. 9b: Hypothalamic gene expression profile of db/db mice
compared to wild type mice. n=6 db/db, n=4 wild type. P values of
significance or strong trend are shown on the bars.
[0111] FIGS. 10a 10e: Hypothalamic gene therapy with an
autoregulatory BDNF vector improved mobility and exploration
behavior of obese db/db mice.
[0112] FIG. 10a: Central distance.
[0113] FIG. 10b: Peripheral distance.
[0114] FIG. 10c: Total distance.
[0115] FIG. 10d: Ratio of central to total distance.
[0116] FIG. 10e: Center time. n=6 YFP, n=5 BDNF-miR-scr, n=9
BDNF-miR-Bdnf. P values are shown on the bars.
[0117] FIGS. 11a-11b: Injection of rAAV-GFPICre vector did not
cause cell loss as shown with Nissl staining (FIG. 11a) or Oasis as
shown by GFAP staining (FIG. 11b). Scale bars: 200 lam.
[0118] FIG. 11c: Double-staining of HA tag and GFP in hypothalamus
of mice 4 months after first surgery (injection of AAV-flax-BDNF)
and 3 months after second surgery (injection of AAV-GFPICre). HA
(left) and GFP (middle) immunoreactivities were found in the same
area of hypothalamus but no colocalization was observed (right).
The majority of cells are GFP immunoreactive with fewer cells
expressing HA consistent with the -72% knockdown of BDNF protein
levels observed in hippocamapal lysates. Scale bar: 50 ttm.
[0119] FIG. 12: Table showing the effects of hypothalamic gene
transfer of BDNF on various biomarkers in serum.
[0120] FIG. 13a: pAM/CBA-NPY-WPRE-BGH plasmid map.
[0121] FIG. 13b: pAM/CBA-NPY-WPRE-BGH nucleotide sequence [SEQ ID
NO:1].
[0122] FIG. 13c: CAG-BDNF-HA-WPRE nucleotide sequence [SEQ ID
NO:2].
[0123] FIG. 14: Two targeting sequences with the highest scores
(Invitrogene RNAi Design Tool) were selected and cloned into the
Block-iT PolII miR RNAi expression vector: WPRE 74:
CTATGTGGACGCTGCTTTA [SEQ ID NO:3], and WPRE155: TCCTGGTTTGTCTCTTTAT
[SEQ ID NO:4]. In in vitro experiments, both miR constructs
inhibited BDNF expression by at least 90% when co-transfected with
the HA-BDNF-WPRE plasmid, as confirmed by ELISA for BDNF.
miR-WPRE74 was chosen to construct the autoregulatory plasmid
shown.
[0124] FIG. 15: The mRNA for human BDNF-nt sequence [SEQ ID NO:5];
aa sequence [SEQ ID NO:10].
[0125] FIG. 16: The mRNA for human trkB-nt sequence [SEQ ID NO:6];
aa sequence [SEQ ID NO: 11].
[0126] FIG. 17: DNA sequence and gene structure of human AGRP nt
sequence [SEQ ID NO:7]; aa sequence [SEQ ID NO: 12].
[0127] FIG. 18: DNA sequence for woodchuck post-transcriptional
regulatory element (WPRE) [SEQ ID NO:8].
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0128] In one broad aspect, there is provided herein a recombinant
adeno-associated virus (rAAV) virion containing a vector expression
cassette having an enhancer and promoter, a regulatory gene
sequence and a poly-A flanked by AAV inverted terminal repeats
(ITR), and having a biologically active protein cDNA fused at the
5' terminal. It is to be understood that, in certain embodiments,
the AAV vector genome can be single stranded containing the ITRs
which flank the genome; and in other embodiments, can be double
stranded so-called "self-complementary" (sc)AAV which also have
ITRs flanking the genome by one ITR which is altered. In one
embodiment, there is a deletion in the D-region of one of the ITRs
which prevents rep-mediated nicking of the newly synthesized rAAV
genome enabling efficient production and packaging of dimeric,
double-stranded rAAV genomes into recombinant sc particles. The
rAAV virion is inserted to one or more cloning sites between the
promoter and the regulatory sequence.
[0129] In certain embodiments, the biologically active protein
comprises a nucleic acid sequence encoding BDNF, or a derivative or
functional fragment thereof, that is expressed in a target cell
either constitutively or under regulatable conditions. In a
particular embodiment, the biologically active protein comprises a
human BDNF protein sequence.
[0130] In another broad aspect, there is provided herein a
pharmaceutical composition comprising the AAV gene therapy particle
in a biocompatible pharmaceutical carrier.
[0131] In a particular aspect, the gene transfer comprises
stereotactic surgery of AAV-BDNF, a recombinant defective AAV virus
to deliver a BDNF cDNA.
[0132] In another particular aspect, there is provided herein an
inventive method that demonstrates a remarkable efficacy on
lowering body weight. Also, there is described herein an inventive
method for altering a metabolic profile in a subject in need
thereof. In certain embodiments, the altered metabolic profile
includes an increase in insulin sensitivity, reduced leptin, and
other changes resembling that of an enriched environment.
[0133] In another particular aspect, AAV mediated BDNF gene
transfer to the hypothalamus leads to an improved metabolic state
with weight loss and biochemical markers consistent with markedly
improved glucose tolerance and reduced fat mass.
[0134] In still another aspect, there is provided herein a
hypothalamic BDNF gene therapy that can be useful as a treatment
for obesity and related metabolic disorders.
[0135] In another broad aspect, there is provided herein a method
of gene therapy for the treatment of a subject having a mutation in
the BDNF gene comprising, administering a therapeutically effective
amount of a recombinant adenoviral associated virus (rAAV) gene
therapy particle to cells of the subject, wherein the gene therapy
particle. In certain embodiments, the AAV gene therapy vector is
administered by a stereotactic route. In a particular embodiment,
the cells of the subject are brain hypothalamic cells. In one
particular embodiment, the cells of the subject are hypothalamic
cells responsible for some or partial control of food intake and/or
the body's metabolism (e.g., the body's metabolic rate and/or
energy burning). Also, in certain embodiments, the subject is a
primate, including, but not limited to, a human.
[0136] In still another aspect, there is provided herein a method
of reducing or eliminating metabolic-related disorder symptoms
comprising administering to a subject in need thereof a
therapeutically effective amount of a recombinant adenoviral
associated virus (rAAV) gene therapy particle as described herein.
In certain embodiments, the metabolic symptoms can be one or more
of obesity, insulin sensitivity, syndrome X, and diabetes.
[0137] In certain embodiments, the promoter of the transgene is a
constitutive promoter. In other embodiments, cellular or hybrid
promoters which may also be responsive to the pathophysiological
state can be used. It is to be understood that, in certain
embodiments, when the target weight is reached, the physiological
responsive promoter might be stronger than the promoter driving the
transgene.
[0138] When the transgene overexpression leads to physiological
changes, the weaker promoter controlling interfering RNA expression
will be activated and thereby induces RNAi to inhibit the transgene
expression. This system can provide a physiological negative
feedback for all gene transfer studies and application in vivo
and/or in vitro.
[0139] In a broad aspect, there is provided herein compositions and
methods of a metabolic disorder treatment utilizing transgene
expression from a rAAV vector containing a BDNF cDNA. Brain-derived
neurotrophic factor (BDNF) is a neurotrophic factor found in the
brain and the periphery. BDNF is a protein that acts on certain
neurons of the central nervous system and the peripheral nervous
system.
[0140] As used herein, the term "adeno-associated virus (AAV)
vector," "AAV gene therapy vector," and "gene therapy vector" refer
to a vector having functional or partly functional ITR sequences
and transgenes. As used herein, the term "ITR" refers to inverted
terminal repeats (ITR).
[0141] Adeno-associated viral vectors (AAV) can be constructed
using known techniques to provide at least the operatively linked
components of control elements including a transcriptional
initiation region, an exogenous nucleic acid molecule, a
transcriptional termination region and at least one
post-transcriptional regulatory sequence. The control elements are
selected to be functional in the targeted cell. The resulting
construct which contains the operatively linked components is
flanked at the 5' and 3' region with functional AAV ITR
sequences.
[0142] Throughout this application, various publications are
referred to by citations within parentheses and in the
bibliographic description, immediately preceding the claims. The
disclosures of these publications are hereby incorporated by
reference into the present disclosure to more fully describe the
state of the art to which this invention pertains, including, but
not limited to: [0143] Kaplitt et al., U.S. Pub. No. 2007/0059290
"Transcriptional regulation of target genes;" [0144] During U.S.,
Pub. No. 2005/0163756 "Oral Delivery of Adeno-Associated Viral
Vectors;" [0145] During U.S., Pub. No. 2005/0136,036 "Methods and
compositions for the Treatment of Neurological Disease;" [0146]
During, U.S. Pub. No. 2004/0131596 "Method and compositions for
modifying target receptor function associated with neurological
disorders;" [0147] During U.S., Pub. No. 2005/0107320 "Methods and
compositions for use in interventional pharmacogenomics;" [0148]
Kaplitt et al., U.S. Pub. No. 2003/0087264 "Transcriptional
regulation of target genes;" [0149] Kaplitt et al., U.S. Pat. No.
6,503,888 "AAV-mediated delivery of DNA to cells of the nervous
system;" [0150] Kaplitt et al., U.S. Pat. No. 6,436,708 "Delivery
system for gene therapy to the brain;" and [0151] Kaplitt et al.,
U.S. Pat. No. 6,180,613 "AAV-mediated delivery of DNA to cells of
the nervous system."
[0152] In another aspect, there is provided herein an AAV gene
therapy particle. Further disclosed herein are pharmaceutical
compositions and methods for treating, preventing or reducing
symptoms of metabolic-related disorders.
[0153] As used herein, the terms "gene transfer," "gene delivery,"
and "gene transduction" can refer to methods or systems for
reliably inserting a particular nucleotide sequence (e.g., DNA)
into targeted cells. As used herein, the term "gene therapy" can
refer to a method of treating a patient wherein polypeptides or
nucleic acid sequences are transferred into cells of a patient such
that activity and/or the expression of a particular molecule is
restored.
[0154] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. All publications,
including patents and non-patent literature, referred to in this
specification are expressly incorporated by reference herein.
DEFINITIONS
[0155] As used herein, the term "gene" refers to an assembly of
nucleotides that encodes a polypeptide and includes cDNA and
genomic DNA nucleic acids. A gene is a nucleic acid that does not
necessarily correspond to the naturally occurring gene which
contains all of the introns and regulatory sequences, e.g.,
promoters, present in the natural genomic DNA. Rather, a gene
encoding a particular protein can minimally contain just the
corresponding coding sequence for the protein.
[0156] As used herein, a "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded at its 3' terminus by the transcription initiation site
and extends upstream (5' direction) to include the minimum number
of bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence will be
found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0157] As used herein, transcriptional and translational control
sequences are DNA regulatory sequences, such as promoters,
enhancers, terminators, and the like, that provide for the
expression of a coding sequence in a host cell. In eukaryotic
cells, polyadenylation signals are control sequences.
[0158] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "operatively under the control"
of transcriptional and translational control sequences in a cell
when RNA polymerase transcribes the coding sequence into a
precursor RNA, which is then trans-RNA spliced to yield mRNA and
translated into the protein encoded by the coding sequence.
[0159] A nucleotide sequence is "operatively under the control" of
a genetic regulatory sequence when the genetic regulatory sequence
controls and/or regulates the transcription of that nucleotide
sequence. That genetic regulatory sequence can also be referred to
as being "operatively linked" to that nucleotide sequence.
[0160] As used herein, a "genetic regulatory sequence" is a nucleic
acid that: (a) acts in cis to control and/or regulate the
transcription of a nucleotide sequence, and (b) can be acted upon
in trans by a regulatory stimulus to promote and/or inhibit the
transcription of the nucleotide sequence. Therefore, an inducible
promoter is a genetic regulatory sequence. In addition, a portion
of a promoter (e.g., fragment/element) that retains and/or
possesses the ability to control and/or regulate the transcription
of a nucleotide sequence either alone or in conjunction with an
alternative promoter or fragment thereof (e.g., a chimeric
promoter), is also a genetic regulatory sequence. Such fragments
include response elements (genetic response elements) and promoter
elements
[0161] As used herein, an "expression cassette" is a nucleic acid
that minimally comprises a nucleotide sequence to be transcribed
(e.g., a coding sequence) that is operatively under the control of
a genetic regulatory sequence.
[0162] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0163] As used herein, a "heterologous gene" is a gene that has
been placed into a vector or cell that does not naturally occur in
that vector or cell.
[0164] As used herein, a gene is an "exogenous gene" when the gene
is not encoded by the particular vector or cell.
[0165] A "vector" as used herein is a genetic construct that
facilitates the efficient transfer of a nucleic acid (e.g., a gene)
to a cell. The use of a vector can also facilitate the
transcription and/or expression of that nucleic acid in that cell.
Non-limiting examples of vectors include plasmids, phages,
amplicons, viruses and cosmids, to which another DNA segment may be
attached so as to bring about the replication of the attached
segment.
[0166] The term "subject" as used herein refers to any living
organism in which an immune response is elicited. The term subject
includes, but is not limited to, humans, nonhuman primates such as
chimpanzees and other apes and monkey species; farm animals such as
cattle, sheep, pigs, goats and horses; domestic mammals such as
dogs and cats; laboratory animals including rodents such as mice,
rats and guinea pigs, and the like. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be covered.
[0167] The term "mammal" as used herein refers to a living organism
capable of eliciting a humoral immune response to an antigen. The
term subject includes, but is not limited to, nonhuman primates
such as chimpanzees and other apes and monkey species, sheep, pigs,
goats, horses, dogs, cats, mice, rats and guinea pigs, and the
like.
[0168] Gene Therapy
[0169] The genetic regulatory sequences can be used to modulate
gene transcription in any cell, including human cells. However, the
genetic regulatory sequences can be used to modulate gene
transcription in cells of other mammals, such as rodents, e.g.,
mice, rats, rabbits, hamsters and guinea pigs; farm animals, e.g.,
sheep, goats, pigs, horses and cows; domestic pets such as cats and
dogs, higher primates such as monkeys, and the great apes such as
baboons, chimpanzees and gorillas. In certain embodiments, the
genetic regulatory sequences can be operatively linked to any
heterologous nucleic acid of interest, preferably those encoding
proteins.
[0170] In addition, the expression cassettes can be constructed to
comprise multiple nucleic acids each encoding a different protein
and all under the control of the same genetic regulatory sequence.
Alternatively, different nucleic acids can be placed under the
control of different genetic regulatory sequences. For example, the
use of two genetic regulatory sequences, one of which stimulates
transcription and the other which hinders transcription under the
same conditions, can be used to control the expression of two
different genes at the same time by operatively linking one coding
sequence to one genetic regulatory sequence and the other coding
sequence to the other genetic regulatory sequence. Alternatively,
multiple expression cassettes can be employed encoding multiple
different proteins. The vectors can be delivered in vitro, ex vivo
and in vivo.
[0171] When the genetic regulatory sequence is contained in a viral
vector, the delivery can be performed by stereotaxic injection into
the brain, for example, as previously exemplified (U.S. Pat. No.
6,180,613, herein specifically incorporated by reference in its
entirety); or via a guide catheter (U.S. Pat. No. 6,162,796, herein
specifically incorporated by reference in its entirety) to an
artery to treat the heart. In certain other embodiments, the
vectors may also be delivered intravenously,
intracerebroventricularly and/or intrathecally, for specific
applications. Additional routes of administration can be local
application of the vector under direct visualization, e.g.,
superficial cortical application, or other non-stereotactic
applications.
[0172] For targeting a vector to a particular type of cell, it may
be necessary to associate the vector with a homing agent that binds
specifically to a surface receptor of the cell. Thus, the vector
may be conjugated to a ligand (e.g., enkephalin) for which certain
nervous system cells have receptors, or a surface specific
antibody. The conjugation may be covalent, e.g., a crosslinking
agent such as glutaraldehyde, or noncovalent, e.g., the binding of
an avidinated ligand to a biotinylated vector. In addition, the
helper-free defective viral vectors of the present invention can be
delivered ex vivo, as exemplified by Anderson et al. (U.S. Pat. No.
5,399,346, herein specifically incorporated by reference in its
entirety).
[0173] Alternatively, a vector can be introduced by lipofection.
Liposomes can be used for encapsulation and transfection of nucleic
acids. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7417; see also Mackey et al. (1988), Proc. Natl. Acad. Sci.
U.S.A 85:8027-8031). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Felgner et al.
(1989) Science 337:387-388). The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as the
brain. Lipids may be chemically coupled to other molecules for the
purpose of targeting (Mackey et. al. (1988) supra).
[0174] In an ex vivo method of the invention, the genetic
regulatory sequences are delivered to a host cell to be
transplanted into a mammalian recipient. The host cells may be
endogenous or exogenous to the mammalian recipient. The term
"transplant cell" refers broadly to the component, e.g., tissue or
cells, being grafted, implanted, or transplanted into a recipient
subject. As used herein, the term "transplantation" refers to the
transfer or grafting of tissues or cells from one part of a subject
to another part of the same subject or to another subject.
Transplanted tissue may comprise a collection of cells of identical
composition, or derived from a donor organism, or from an in vitro
culture. Delivery of the genetic regulatory sequences of the
invention to a transplant cell may be accomplished by any of the
methods known to the art and described herein, e.g., as a plasmid,
as part of a vector; by injection, lipofection, etc.
[0175] Transgenic Animals
[0176] A transgenic animal model can be prepared so as to contain a
nucleic acid operatively under the control of a genetic regulatory
sequence of the present invention. For example, transgenic vectors,
including viral vectors, or cosmid clones (or phage clones) can be
constructed. Cosmids may be introduced into transgenic mice using
published procedures (Jaenisch (1988) Science 240:1468-1474).
[0177] Thus, the present invention further provides transgenic,
knock-in, and knockout animals that contain one or more
heterologous genes operatively under the control of a genetic
regulatory sequence of the present invention. These animals can be
used as animal models in drug screening assays. In one such
example, a drug can be added under various "controlled" expression
levels of a particular gene, or at various time points before
and/or after induced expression of the particular gene, allowing a
much more detailed investigation of the effects of that drug on a
particular condition. In a specific embodiment, the transgenic,
knock-in, or knockout animal is a mouse. Cells from the inducible
knockout, knock-in and/or transgenic animals of the present
invention are also part of the present invention.
[0178] Transgenic animals can be obtained through gene therapy
techniques described above or by microinjection of a nucleic acid,
for example, into an embryonic stem cell or an animal zygote (such
as a bacterial artificial chromosome (BAC) comprising a nucleic
acid operatively under the control of a genetic regulatory sequence
of the present invention). Microinjection of BACs has been shown to
be successful in a number of animals including rats, rabbits, pigs,
goats, sheep, and cows (in Transgenic Animals Generation and Use
(1997) ed., L. M. Houdebine, Harwood Academic Publishers, The
Netherlands). Methods of constructing BACs or other DNAs such as
bacteriophage P1 derived artificial chromosomes (PACs) that encode
specific nucleic acids through homologous recombination have
recently been described in great detail (Heintz et al. (1998)
PCT/US98/12966, herein specifically incorporated by reference in
its entirety). Alternatively, a yeast artificial chromosome (YAC)
can be used.
[0179] Ribozymes and Antisense
[0180] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(see Weintraub (1990) Sci. Amer. 262:40-46; Marcus-Sekura (1987)
Nucl. Acid Res, 15:5749-5763; Marcus-Sekura (1988) Anal. Biochem.
172:289-295); Brysch et al. (1994) Cell Mol. Neurobiol.
14:557-568). Preferably, the antisense molecule employed is
complementary to a substantial portion of the mRNA. In the cell,
the antisense molecule hybridizes to that mRNA, forming a double
stranded molecule. The cell does not translate an mRNA in this
double-stranded form. Therefore, antisense nucleic acids interfere
with the expression of mRNA into protein. Preferably a DNA
antisense nucleic acid is employed since such an RNA/DNA duplex is
a preferred substrate for RNase H. Oligomers of greater than about
fifteen nucleotides and molecules that hybridize to the AUG
initiation codon will be particularly efficient. Antisense methods
have been used to inhibit the expression of many genes in vitro
(Marcus-Sekura (1988) supra; Hambor et al. (1988) Proc. Natl. Acad.
Sci. U.S.A. 85:4010-4014) and in situ (Arima et al. (1998)
Antisense Nucl. Acid Drug Dev. 8:319-327; Hou et al. (1998)
Antisense Nucl. Acid Drug Dev. 8:295-308).
[0181] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA molecules in a manner
somewhat analogous to DNA restriction endonucleases. Ribozymes were
discovered from the observation that certain mRNAs have the ability
to excise their own introns. By modifying the nucleotide sequence
of these ribozymes, researchers have been able to engineer
molecules that recognize specific nucleotide sequences in an RNA
molecule and cleave it (Cech (1988) JAMA 260:3030-3034; Cech (1989)
Biochem. Intl. 18:7-14). Because they are sequence-specific, only
mRNAs with particular sequences are inactivated.
[0182] Pharmaceuticals
[0183] Pharmaceutical compositions may be administered alone or in
combination with at least one other agent, such as a stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier, including, but not limited to, saline,
buffered saline, dextrose, and water. The compositions may be
administered to a patient alone, or in combination with other
agents, modulators, or drugs (e.g., antibiotics).
[0184] In particular embodiments, the pharmaceutical compositions
also contain a pharmaceutically acceptable carrier or excipient.
Such materials should be non-toxic and should not interfere with
the efficacy of the active ingredient. Pharmaceutically acceptable
excipients include, but are not limited to, liquids such as water,
saline, glycerol, sugars and ethanol. Pharmaceutically acceptable
salts can also be included therein, for example, mineral acid salts
such as hydrochlorides, hydrobromides, phosphates, sulfates, and
the like; and the salts of organic acids such as acetates,
propionates, malonates, benzoates, and the like. Additionally,
auxiliary substances, such as wetting or emulsifying agents, pH
buffering substances, and the like, may be present in such
vehicles. A thorough discussion of pharmaceutically acceptable
excipients is available in Remington's Pharmaceutical Sciences
[Mack Pub. Co., 18th Edition, Easton, Pa. (1990)]. The precise
nature of the carrier or other material may depend on the route of
administration. As described herein, the present invention is
directed to administering the expression vectors and compositions
thereof of the invention to target cells in the nervous system.
[0185] In accordance with the present invention, an expression
vector that is to be given to an individual, is administered
preferably in a "therapeutically effective amount" or a
"prophylactically effective amount" (as the case may be, although
prophylaxis may be considered therapy), this being sufficient to
show benefit to the individual.
[0186] Although the compositions of the invention have been
described with respect to human therapeutics, it will be apparent
to one skilled in the art that these tools are also useful in
animal experimentation directed to developing treatment regimens
for animal subjects that have a neurological disorder. Indeed, as
described herein, animal subjects which exhibit symptoms
characteristic of various 1 disorders have been developed that
serve as model systems for such human disorders.
SPECIFIC EMBODIMENTS
[0187] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
Example I
Methods
[0188] Mice. We used male 8-week-old C57BL/6 mice (from Charles
River) and male 4-month-old db/db mice (from Jackson Laboratories).
All use of animals was approved by and in accordance with the Ohio
State University Animal Care and Use Committee.
[0189] Recombinant adeno-associated viral vector construction and
packaging. The rAAV plasmid contains a vector expression cassette
consisting of the cytomegalovirus enhancer, the chicken
.beta.-actin (CBA) promoter, the woodchuck post-transcriptional
regulatory element (WPRE) and bovine growth hormone polyadenosine
flanked by AAV inverted terminal repeats. We fused human BDNF cDNA
to the HA tag at the 5' terminus and then inserted it into the
multiple cloning sites between the CBA promoter and the WPRE
sequence. We cloned the genes encoding EGFP or destabilized YFP
into the rAAV plasmid as controls. We packaged and purified rAAV
serotype 1 vectors.
[0190] Adeno-associated virus-mediated brain-derived neurotrophic
factor overexpression in mice kept on standard diet. We randomly
assigned 23 C57BL/6 mice, male, 8 weeks of age, to groups receiving
rAAV-BDNF (n=13) or rAAV-GFP (n=10). We anesthetized the mice with
a single dose of ketamine and xylazine (100 mg kg-1 and 20 mg kg-1,
respectively, intraperitoneally) and secured them via ear bars and
an incisor bar on a Kopf stereotaxic frame. We made a midline
incision through the scalp to reveal the skull and drilled two
small holes into the skull with a dental drill above the injection
sites (1.2 mm posterior to the bregma, 0.5 mm lateral to the
midline, 6.2 mm dorsal to the bregma). We injected rAAV vectors
(3.times.10.sup.9 genomic particles per site) bilaterally into the
hypothalamus at a rate of 0.1 .mu.l min-1 with a 10-.mu.l Hamilton
syringe attached to a Micro4 Micro Syringe Pump Controller (World
Precision Instruments). At the end of infusion, we slowly raised
the syringe from the brain and sutured the scalp. We placed the
mice back into a clean cage and carefully monitored them after
surgery until recovery from anesthesia. We fed mice with normal
chow diet (NCD, 11% fat, 28% protein, 61% carbohydrate, caloric
density 3.4 kcal g-1, Research Diets).
[0191] High-fat diet-induced obesity model. We randomly assigned 24
male C57BL/6 mice, 18 weeks of age, to groups receiving rAAV-BDNF
(n=13) or rAAV-YFP (n=11). We injected rAAV vectors (2.times.109
genomic particles per site) bilaterally into the hypothalamus as
described above. We switched the diet to high-fat diet (HFD, 45%
fat, caloric density 4.73 kcal g-1, Research Diets) on day 10 after
rAAV injection and fed the mice with HFD until the end of the study
(72 d after injection).
[0192] microRNA vector construction and adeno-associated vector
production. We used microRNA to target BDNF. We cloned two
targeting sequences in the BDNF coding region into the Block-iT
PolII miR RNAi expression vector (pcDNA6.2-Gw/miR, Invitrogen). In
in vitro experiments, both miR constructs inhibited BDNF expression
when co-transfected with a BDNF expression plasmid, as confirmed by
quantitative PCR and ELISA for BDNF (BDNF Emax ImmunoAssay System,
Promega). We chose the miR-Bdnf construct with mature microRNA
sequence: 5'-AATACTGTCACACACGCTCAG-3') [SEQ ID NO:9] for in vivo
experiments. We subcloned this miR-Bdnf and a scrambled microRNA
(miR-scr, with the scrambled sequence targeting no known gene,
Invitrogen) into the rAAV plasmid driven by CBA promoter as
described herein.
[0193] Auto-regulatory system. We amplified two AGRP promoter
fragments from human genomic DNA by PCR. FIG. 17 shows the DNA
sequence and gene structure of human AGRP [SEQ ID NO:7].
[0194] AGRP484 (484 bp, -133 bp to +351 bp from the start of the
noncoding exon28) [see SEQ ID NO:8], and AGRP814 (814 bp,
-463/+351) [see SEQ ID NO:8].
[0195] We inserted the AGRP promoter fragments into rAAV vectors to
drive luciferase report gene expression. To verify the induction of
AGPR promoter by BDNF-induced physiological change, we injected the
combination of viral vectors into the arcuate nucleus of four
groups of mice: those expressing YFP and AGRP484-luc, those
expressing YFP and AGRP814-luc, those expressing BDNF and
AGRP484-luc and those expressing BDNF and AGRP814-luc. We injected
an equal amount of each viral vector (1.5.times.10.sup.9 genomic
titer) bilaterally. We killed the mice and dissected the
hypothalamus 3 weeks after rAAV injection. We measured the
luciferase activity in the hypothalamic lysate by Bright-Glo
Luciferase Assay (Promega) and calibrated the luminescence to the
protein concentration. We chose AGRP484 to develop the
autoregulatory system. We generated vectors containing two
cassettes, one cassette expressing BDNF driven by the CBA promoter
as described above and the other cassette expressing microRNA
(miR-Bdnf or miR-scr) driven by AGRP484. Both the transgene and the
microRNA cassettes were packaged to a single viral vector
(BDNF-miR-scr, BDNF-miR-Bdnf).
[0196] db/db mice. We randomly assigned 30 db/db mice to groups
receiving rAAV-YFP, rAAV-BDNF-miR-scr or rAAV-BDNF-miR-Bdnf, with
ten mice per group. We injected rAAV vectors (3.4.times.10.sup.10
genomic particles per site) bilaterally into the hypothalamus as
described above. We fed the db/db mice with NCD throughout the
experiment. We killed the mice receiving rAAV-BDNF-miR-scr one
month after injection owing to their severe weight loss. We
recorded the body weight and food consumptions periodically until
the end of the experiment (79 d after injection).
[0197] Knockdown of transgene expression by Cre-loxP recombination.
We generated the DIO model by feeding mice with HFD for 10 weeks
until the body weight reached 40 g. We randomly assigned the obese
mice to groups receiving rAAV flox-BDNF or rAAV-YFP. We injected
rAAV vectors bilaterally to the hypothalamus as described above
(1.0.times.10.sup.10 genomic particles per site). We monitored body
weight every 5-7 d and recorded the food intake. One month after
first surgery, we split the flox-BDNF-expressing mice into two
groups receiving rAAV-GFP-Cre or empty rAAV as a control. We
injected all YFP-expressing mice with rAAV-GFP-Cre. We performed
the second surgery with the same procedure as the first surgery. We
kept the mice on HFD until the end of the study (4 months after the
first surgery).
[0198] Statistical analyses. Values are expressed as
means.+-.s.e.m. For body weight, insulin tolerance and glucose
tolerance, we determined the overall significance by one-way
repeated measure analysis of variance. We used one-way analysis of
variance to analyze serum biomarker measurements, liver weight and
adipose tissue weight. We used multivariate analysis of variance to
analyze quantitative RT-PCR data.
[0199] Body weight and food consumption. We maintained the mice on
a normal 12 h/12 h light/dark cycle with respective diet (NCD or
HFD) and water ad libitum throughout the experiment. Body weight of
each individual mouse was recorded before injection and every 3-7
days after injection. Food consumption was recorded periodically
after injection as the total food consumption of each cage housing
4-5 mice and represented as the average of food consumption per
mouse per day.
[0200] Serum harvest and biomarker measurement. We collected blood
from the retroorbital sinus 3-4 weeks after AAV injection. We
anesthetized the mice of each group at the same time with ketamine
(87 mg kg-1)/xylazine (13 mg kg-1) followed by blood withdraw. All
blood harvesting started at 10:00 am. We prepared serum by allowing
the blood to clot for 30 min on ice followed by centrifugation.
Serum was at least diluted 1:5 in serum assay diluent and assayed
using the following DuoSet ELISA Development System (R&D
Systems): mouse IGF-1, IGFBP-3, Leptin, Leptin R,
Adiponectin/Acrp30. Insulin was measured using Mercodia
ultrasensitive mouse insulin ELISA (ALPCO Diagnostic). Glucose was
measured using QuantiChrom Glucose Assay (BioAssay Systems). Total
cholesterol was measured using Cholesterol E test kit (Wako
Diagnostics). Triglyceride was measured using L-Type test (Wako
Diagnostics).
[0201] BDNF expression quantification. We dissected hypothalami and
prepared total RNA from half of the hypothalamic tissue and
subjected it to quantitative RT-PCR. We calibrated the data of
quantitative RT-PCR to the endogenous control gene Eef2. We
prepared lysates from the other half of the hypothalamic tissue and
measured BDNF protein level using ELISA (BDNF Emax ImmunoAssay
System, Promega). The BDNF protein level was calibrated to the
total protein level.
[0202] Red-O staining. We stained lipids in liver and white adipose
tissue frozen sections using an Oil Red-O solution (Sigma).
[0203] Glucose tolerance test. We injected mice intraperitoneally
with glucose solution (1 mg glucose per kg body weight) after an
overnight fast. We obtained blood from the tail at various time
points. We measured blood glucose concentrations with a portable
glucose meter (ReliOn Ultima).
[0204] Insulin tolerance test. We injected mice intraperitoneally
with insulin (0.75 unit per kg body weight) at 2 pm without a fast.
We obtained blood from the tail and measured the blood glucose
concentration as described herein.
[0205] Quantitative RT-PCR. We dissected liver, white adipose
tissue and hypothalamus and isolated total RNA using RNeasy Mini
Kit plus RNase-free DNase treatment (Qiagen). We generated
first-strand cDNA using TaqMan Reverse Transcription Reagent
(Applied Biosystems) and carried out quantitative PCR using an ABI
PRISM 7000 Sequence Detection System with the Power SYBR Green PCR
Master Mix (Applied Biosystems). We designed primers to detect the
following mouse mRNA: Bdnf, Npy, Agrp, Sgk1, Vgf, Insr, Lepr,
Ntrk2, Cartpt, Pomc, Mc4r, Trh, Crh, Ucp1, Ucp2, Ucp3, Lep, Adipoq,
Cycs, Fasn, Ppargc1a, Rb1, Pparg, Dio2, Acox1, Cpt1a, Gpam, Scd1,
Srebf1. Primer sequences are available on request. We calibrated
data to endogenous control Actb in liver and adipose tissue, Eef2
in hypothalamus and quantified the relative gene expression using
the equation T0/R0=K.times.2 (CT,R-CT,T). T0 is the initial number
of target gene mRNA copies, R0 is the initial number of internal
control gene mRNA copies, CT,T is the threshold cycle of the target
gene, CT,R is the threshold cycle of the internal control gene and
K is a constant.
[0206] rAAV-microRNA experiment. We randomly assigned 7 week old
C57/BL6 mice to receive AAV-CBA-miR-Bdnf (n=10) or AAV-CBA-miR-scr
(n=10). We injected 0.7 .mu.l of AAV vectors (1.4.times.10.sup.10
particles) bilaterally into the hypothalamus at the stereotaxic
coordinates described above. We sacrificed the mice 30 days after
vector injection and dissected the hypothalamus for Q-PCR and BDNF
ELISA.
[0207] Immunohistochemistry. We perfused mice with 20 ml cold PBS
followed by 50 ml 4% paraformaldehyde. Coronal brain sections (20
.mu.m) were cut using a Leica freezing microtome and
immunofluorescence staining was performed with the following
antibodies: monoclonal antibody to HA tag (Covance, 1:250) followed
by Cy3conjugated secondary antibody (Jackson Immunoresearch,
1:400); polyclonal antibody to NPY (Chemicon, 1:8,000) followed by
DyLight488-conjugated secondary antibody (Jackson Immunoresearch,
1:600); polyclonal antibody to GFAP (Dako, 1:500) followed by
Cy3-conjugated secondary antibody (Jackson Immunoresearch, 1:400).
Apoptosis was assessed by TUNEL assay using in situ Cell Death
Detection Kit (Fluorescence, Roche) according to manufacturer's
instruction and counterstained with DAPI. We detected
immunofluorescence with Zeiss Axioskop40 microscope and took
pictures and processed the pictures with Zeiss AxioVision3.1
software. We processed confocal laser scanning with Zeiss 510 Meta
Laser Scanning Confocal microscope.
[0208] Open field. To assess exploration and general motor
activity, we placed mice individually into the center of an open
square arena (60 cm.times.60 cm, enclosed by walls of 48 cm). The
mouse was allowed 10 min in the arena, during which time its
activity was recorded and analyzed by Clever Systems TopScan
Software (Clever Sys Inc, Vienna, Va.). Specifically, we measured
the distance traveled both in the center of the arena (36
cm.times.36 cm), the total distance traveled and the time spent in
the center of the arena. The total distance traveled provides a
measure of exploratory activity while the time and distance ratio
of arena center exploration provides a preliminary indication of
anxiety level. We cleaned the arena with 30% ethanol between trials
to remove any odor cues.
[0209] Flox-BDNF vector. We generated flox-BDNF plasmid by
inserting two lox P flanking the human BDNF-HA cDNA in the AAV
vector. We packaged the following rAAV1 viral vectors: flox-BDNF,
Cre recombinase fused to GFP, and empty vectors as control.
[0210] Metabolic studies. We measured energy expenditure and
activity of mice using the Oxymax Lab Animal Monitoring System
(Columbus Instruments). Individual mice were allowed to be
habituated to the instrument overnight and the physiological and
behavioral parameters were monitored for 24 h (activity, food and
water consumption, metabolic performance and temperature). Oxygen
consumption, carbon dioxide production and methane production were
normalized to the body weight and corrected to an effective mass
value according to the manufacturer's software.
[0211] Bone mineral density. We measured the volumetric bone
mineral density (vBMD) by microcomputed tomography (.mu.CT, Siemens
Invion, Wright Center for Innovation, The Ohio State
University).
[0212] Results for Example I
[0213] Hypothalamic Gene Transfer of Bdnf in Normal Mice Fed a
NCD
[0214] We delivered hemagglutinin (HA)-tagged human BDNF to the
hypothalamus bilaterally via rAAV, using GFP as a control. In a
subset of mice, we determined gene transfer efficacy by HA
immunofluorescence and GFP fluorescence for the respective vectors,
with expression observed in the arcuate nucleus and ventromedial
hypothalamus (FIGS. 1a,b). Consistent with the use of a
constitutive promoter, we observed expression in the majority of
neurons in the targeted region, including in arcuate agouti-related
protein (AgRP) and neuropeptide Y (NPY) coexpressing neurons (FIG.
1c). We observed an initial surgery-associated weight loss in both
groups, but GFP-expressing mice quickly recovered and then regained
weight on their presurgery trajectory (FIG. 1d). In contrast,
BDNF-expressing mice continued to lose weight throughout the course
of the experiment (FIG. 1d). By one month after injection, the
weight of BDNF-expressing mice had decreased by 3.66.+-.0.27 g,
whereas the weight of GFP-expressing mice had increased by
1.91.+-.0.37 g. There was no significant change in food consumption
(FIG. 6a). Adiposity was greatly reduced in BDNF-expressing mice,
as indicated by a 92% reduction in the weight of the perigonadal
fat pad at 50 d after injection (FIG. 1e).
[0215] BDNF expression led to a sharp decrease in leptin abundance
(12.2%.+-.2.6% of GFP-expressing mice, P<0.001) and insulin
(18.0%.+-.2.3% of GFP, P<0.001). Both leptin and insulin
concentrations are known to correlate with fat mass. Moreover,
expression of adiponectin, a major adipokine with a role in
regulating insulin sensitivity and inhibiting appetite, was
markedly increased in BDNF-expressing mice, whereas cholesterol,
triglyceride and insulin-like growth factor-1 concentrations were
all reduced (FIG. 12).
[0216] We used real-time quantitative PCR to examine hypothalamic
expression of genes involved in energy homeostasis. Agrp and Npy,
which encode two orexigenic peptide hormones, were upregulated
15.11.+-.1.44-fold and 7.55.+-.1.04-fold in BDNF-expressing,
compared to GFP-expressing, mice, respectively (FIG. 1f),
consistent with a compensatory response to the weight loss and fat
depletion. Mc4r (encoding melanocortin-4 receptor), proposed to be
upstream of both BDNF and a major pathway shared by leptin, insulin
and other anorexic signals, was upregulated significantly, whereas
expression levels of the additional anorexigenic molecules Cartpt
(encoding CART prepropeptide) and Pomc (encoding
proopiomelanocortin) were not changed (FIG. 1f). Expression of the
BDNF receptor Ntrk2 was increased, indicating positive feedback
(FIG. 1f). Insulin receptor (Insr) expression was also upregulated,
whereas expression of the leptin receptor long form (Lepr) was not
changed (FIG. 1f). Expression of Trh (encoding
thyrotropin-releasing hormone) and Crh (encoding
corticotropin-releasing hormone) was increased in BDNF-expressing
mice (FIG. 1f).
[0217] BDNF Gene Transfer Prevents Diet Induce Obesity (DIO)
[0218] Chronic consumption of a HFD contributes to obesity in
experimental animals and humans. We used C57BL/6 mice, a strain
prone to DIO, to assess the therapeutic efficacy of hypothalamic
BDNF gene transfer. Given the potency of the rAAV-BDNF vector
observed in normal mice, we decreased the dose (from
3.times.10.sup.9 genomic titer per site to 2.times.10.sup.9 genomic
titer per site) and used older (18 weeks) mice. We also used a
destabilized yellow fluorescent protein (YFP) as our control, as
GFP has been associated with nonspecific toxic effects. Ten days
after surgery, we switched the mice to a 45% HFD. The weight gain
of YFP-expressing mice, which acted as our control treatment group,
accelerated, whereas BDNF-expressing mice maintained a stable
weight (FIG. 2a).
[0219] By 72 d after surgery, YFP-expressing mice had gained
13.78.+-.1.88 g on the HFD, whereas BDNF-expressing mice lost
2.80.+-.0.71 g with no change in food consumption (FIG. 6b).
YFP-expressing mice developed abdominal obesity with the weight of
perigonadal fat pads increased by 4.5-fold compared to NCD controls
(data not shown). In contrast, the perigonadal pad weight of
BDNF-expressing mice was only 14.2.+-.3.1% that of the
YFP-expressing mice. The weight of subcutaneous fat was also
greatly less (FIGS. 2b, 2c), and the pericardial fat observed in
YFP-expressing mice was completely absent in the BDNF-expressing
mice (FIG. 2b). Moreover, H&E staining revealed an 85.7.+-.1.1%
smaller adipocyte size in BDNF-expressing mice (FIG. 2d).
[0220] DIO was associated with hyperinsulinemia, hyperleptinemia,
hyperglycemia and dyslipidemia, with BDNF completely preventing
this metabolic profile (FIG. 12). The lower circulating leptin
concentrations were not solely due to the smaller fat mass. When
leptin concentrations were standardized to the perigonadal fat pad
weight, BDNF-expressing mice still showed a significant decrease
(YFP-expressing mice, 7.347.+-.0.612 pg ml-1 g-1; BDNF-expressing
mice, 3.735.+-.0.798 pg ml-1 g-1; P=0.003).
[0221] In contrast, adiponectin showed a greater than 14-fold
higher level when its concentration was corrected to fat mass
(YFP-expressing mice, 1.624.+-.0.314 ng ml-1 g-1; BDNF-expressing
mice, 24.030.+-.5.540 ng ml-1 g-1; P=0.005), indicating that BDNF
expression influenced adipocyte autonomous leptin and adiponectin
secretion. In addition, the insulin insensitivity and glucose
intolerance observed in YFP-expressing obese mice were greatly
improved in BDNF-expressing mice (FIG. 2 and FIG. 6c).
[0222] HFD feeding led to liver steatosis in obese YFP-expressing
mice, as characterized by pale macroscopic enlargement (FIG. 2b)
and excessive fat accumulation observed in oil red O-stained (FIG.
2e) and H&E-stained (FIG. 2f) sections. BDNF expression
prevented the liver steatosis (FIGS. 2e, 2f), with the liver weight
being 45% that of the YFP-expressing mice (BDNF-expressing mice,
1.18.+-.0.05 g; YFP-expressing mice, 2.61.+-.0.29 g; P=0.001).
[0223] We profiled the expression of genes involved in lipid
metabolism and mitochondrial activity in both white adipose tissue
(WAT, FIG. 3a) and brown adipose tissue (data not shown). In WAT,
Ppargc1a, encoding a cofactor controlling mitochondrial biogenesis,
was upregulated 9.8.+-.2.2-fold in BDNF-expressing mice, whereas
the expression of cytochrome c (Cycs) was increased 3.2.+-.0.5-fold
compared to the obese control mice. Uncoupling proteins (UCP) are a
family of proteins involved in the regulation of lipid oxidation,
as well as the regulation of energy expenditure. The expression of
Ucp3 was increased 6.8.+-.1.9-fold in WAT from mice expressing
BDNF, suggesting a possible increase in energy expenditure of WAT.
Moreover, Lep (encoding leptin) expression was decreased by
62.0%.+-.7.9%, whereas Adipoq (encoding adiponectin) expression was
increased by 6.1.+-.1.9-fold, consistent with the observed weight
loss. Lep expression was also decreased by 68.5%.+-.14.3% in BDNF
brown adipose tissue whereas no other genes screened were
significantly changed.
[0224] BDNF treatment significantly suppressed lipogenic gene
expression in liver, decreasing Fasn (encoding fatty acid synthase)
expression by 43.1%.+-.17.5%, Gpam (encoding mitochondrial
glycerol-3-phosphate acyltransferase) by 66.4%.+-.4.2% and Scd1
(encoding stearoyl-CoA desaturase) by 37.8%.+-.10.9%. Its effects
on lipolytic genes were less marked, with a decrease in Acox1
(encoding acyl-coenzyme A oxidase-1, palmitoyl) expression but no
change in Cpt1a (encoding carnitine palmitoyltransferase 1A)
expression, although both are involved in fatty acid oxidation
(FIG. 3b).
[0225] Expression of Pparg, an adipocyte-specific peroxisome
proliferator-activated receptor isoform and a type 2 diabetes
marker, was decreased by 99.6%.+-.0.1% in the liver of BDNF-treated
mice, consistent with the absence of fatty infiltration. Expression
of Ucp2, a mitochondrial inner-membrane protein that uncouples ATP
synthesis and negatively regulates reactive oxygen species
production, was significantly upregulated by 3.39.+-.0.86-fold in
the liver of BDNF-treated mice, which may serve a protective role
when hepatocytes are exposed to metabolic stress such as high-fat
feeding. We also profiled hypothalamic gene expression in
BDNF-expressing mice on a HFD and observed a similar pattern of
changes as BDNF-expressing mice fed with a NCD except that Cartpt
was upregulated approximately threefold in the HFD condition (FIG.
11 versus FIG. 3c).
[0226] Transgene expression in the hypothalamus was maintained
throughout the duration of the experiment, with BDNF concentrations
of 5541.4.+-.738.4 pg mg-1 in BDNF-expressing mice compared to
87.6.+-.11.2 pg mg-1 in YFP-expressing mice, P<0.001. Moreover,
histological examination of hypothalamic sections showed a lack of
cytotoxicity with no cell loss (as determined by Nissl staining,
FIG. 7a), no gliosis (as determined by glial fibrillary acidic
protein staining, FIG. 7b) and no apoptosis (TUNEL assay, FIG.
7d).
[0227] An Autoregulatory BDNF Vector in Diabetic Db/Db Mice
[0228] Gene therapy dose titration in humans is difficult,
particularly in a clinical setting where diet is not tightly
controlled. We, therefore, aimed to improve the safety of the
approach and develop a vector that could be considered for clinical
translation by tightly coupling transgene expression to the
physiological changes induced by the expression of the introduced
therapeutic gene. Of the hypothalamic genes profiled in
BDNF-expressing mice, Agrp was the most robustly upregulated, with
15.1-fold and 16.2-fold increases in expression in mice on NCD and
HFD, respectively, (FIG. 11 and FIG. 3c) consistent with the
observed weight loss and particularly the decrease in body fat
mass.
[0229] We amplified two human AGRP promoter fragments of different
lengths, each containing the hypothalamus-specific exon. We coupled
the AGRP promoter fragments to a luciferase reporter gene and
packaged these cassettes into rAAV vectors. We injected these AGRP
promoter-driven luciferase vectors into the hypothalamus together
with the BDNF vector to induce weight loss and compared luciferase
activity with YFP controls.
[0230] The 484-base pair (bp) fragment promoter (termed AGRP484)
showed better inducibility than the 814-bp fragment. The induction
of AGRP484 was 2.66.+-.0.57-fold, whereas the induction of AGRP814
was 1.32.+-.0.38-fold (n=6 each group), analyzed when
BDNF-expressing mice had lost 1.5.+-.0.22 g of weight and YFP mice
gained 1.5.+-.0.56 g.
[0231] We then used AGRP484 to drive a microRNA targeting BDNF
(FIG. 8), which we inserted into the parent vector containing the
constitutively expressing BDNF complementary DNA cassette. That is,
we made a single vector that expressed both BDNF under the control
of a general constitutive promoter and a microRNA directed against
BDNF under the control of the AGRP promoter (AGRP484-miR-Bdnf), a
promoter that increases activity as the mice lose weight (FIG.
4a).
[0232] Because rAAV-mediated BDNF expression leads to increased
weight loss, the inventors herein now believe that this
physiological event would lead to increased AGRP promoter activity.
That increased AGRP promoter activity, in turn, would drive the
expression of the BDNF-specific microRNA in the same rAAV vector,
resulting in a decrease in BDNF expression. Therefore, a balance
between weight loss and weight gain would be achieved, thus
preventing pathological cachexia from occurring, especially if the
treated mouse's diet is not carefully controlled.
[0233] We used a mouse model of type 2 diabetes, db/db mice, and
delayed the rAAV-mediated treatment until they were extremely obese
and diabetic to investigate both the therapeutic efficacy as well
as the autoregulatory efficiency of the dual-cassette vectors. When
administered a control YFP-encoding virus, db/db mice continued to
gain weight (FIG. 4b).
[0234] In contrast, when the mice were given a BDNF-encoding vector
together with a scrambled microRNA (BDNF-miR-scr, targeting no
known genes), their weight dropped precipitously by 45.3%.+-.3.6%
in 3 weeks (FIG. 4b). The weights of the mice receiving the BDNF
plus the AGRP484-miR-Bdnf dropped markedly but began to level off
and stabilized between 3 and 4 weeks after rAAV injection, with
body weight maintained for the entire 11-week duration of the
experiment, indicating efficient autoregulation of the BDNF
transgene expression (FIGS. 4b, 4c).
[0235] Both BDNF-miR-Bdnf-expressing and BDNF-miR-scr-expressing
mice showed reduced food intake compared to YFP-expressing controls
(FIG. 4d), with increased rectal temperature indicating increased
energy expenditure (data not shown). Moreover, gene therapy with
the autoregulatory BDNF vector alleviated the obesity (FIG. 4e),
improved the insulin sensitivity and glucose tolerance (FIGS. 4f,
4g) and ameliorated the metabolic disturbances in db/db mice (FIG.
4h).
[0236] The profile of hypothalamic gene expression showed a similar
pattern to that observed in wild-type mice but with a milder extent
of changes that is likely to reflect the more controlled BDNF
overexpression (FIG. 9a). Indeed, hypothalamic BDNF levels were
2055.6.+-.402.7 pg mg-1 in BDNF-miR-Bdnf-expressing mice, an 85%
reduction from the 13323.3.+-.3899.8 pg mg-1 concentration in the
BDNF-miR-Scr-expressing mice (P=0.023), and 100.7.+-.13.1 pg mg-1
in YFP-expressing mice. Gene therapy also improved the mobility of
the extremely obese db/db mice and enhanced their physical activity
and exploration behavior, as shown in an open-field test (FIG.
10).
[0237] BDNF-Induced Weight Loss is Reversible by Transgene
Knockout
[0238] To provide a further safeguard for this approach and the
potential for a clinical rescue procedure, we wanted to use the
loxP-Cre recombination system to generate a way to knock out the
transgene, should the need arise because of adverse events.
[0239] We generated a rAAV vector with the BDNF transgene flanked
by two loxP sites (flox-BDNF), which could be subsequently knocked
out by a second viral vector delivering Cre recombinase. The rAAV
vector encoding a GFP-Cre fusion protein has been shown to
efficiently ablate loxP-modified genes in the brain, including the
hypothalamus, with low toxicity. Bilateral injection of Cre vector
alone did not influence body weight (data not shown) and did not
cause toxicity (FIG. 7e and FIGS. 11a, 11b).
[0240] To establish an obesity model with greater clinical
relevance, we fed C57BL/6 mice with the HFD for 10 weeks until
their body weight reached 40 g. The floxed BDNF vector was injected
into the hypothalamus of the obese mice with nonfloxed YFP as a
control (FIG. 5).
[0241] Flox-BDNF-expressing mice started to lose weight 7 d after
injection and by 24 d had lost 29.3%.+-.1.9% of their body weight,
at which time the YFP-expressing mice had gained 9.0%.+-.1.5% of
their baseline weight (FIG. 5a). Food consumption was slightly but
significantly reduced in BDNF-expressing mice (BDNF-expressing
mice, 2.06.+-.0.09 g per mouse per d, YFP-expressing mice,
2.44.+-.0.05 g per mouse per d, P=0.003).
[0242] In addition, energy expenditure (kilocalories of heat
produced) was markedly increased in BDNF-expressing mice during
both the dark phase and light phase (FIG. 5b).
[0243] Physical activity was substantially increased in BDNF mice
by 4.03.+-.0.28-fold compared to YFP mice in a 24 h period,
particularly in the dark phase (FIG. 5c).
[0244] Notably, the respiratory exchange ratio was increased from
0.78.+-.0.03 in YFP-expressing mice to 0.87.+-.0.01 in
BDNF-expressing mice (FIG. 5d), showing increased carbohydrate
oxidation as opposed to lipid oxidation, although both groups were
fed with HFD.
[0245] Obesity-associated glucose intolerance was alleviated by
BDNF treatment 3 weeks after rAAV injection (FIG. 5e).
[0246] We then randomized flox-BDNF-expressing mice to groups
receiving a second viral vector injection to the same site as the
first surgery by receiving either GFP-Cre or empty viral vector as
a control. All YFP-expressing mice received the GFP-Cre viral
vector in the second surgery. GFP-Cre vector injection
significantly suppressed BDNF mRNA and protein expression by
63.9%.+-.9.0% and 71.6%.+-.1.9%, respectively (P=0.002).
Hypothalamic immunohistochemistry showed widespread GFP-Cre
expression with less residual HA immunoreactivity, consistent with
the .about.72% protein knockdown (FIG. 11c).
[0247] Moreover, HA-positive cells (those transduced by flox-BDNF
vector) and GFP-positive cells (those transduced by GFP-Cre vector)
were located in the same area, but no colocalization was observed,
consistent with efficient Cre recombinase activity in co-transduced
cells (FIG. 11c). After the second surgery, YFP-expressing mice
continued to gain weight, whereas flox-BDNF-expressing mice
receiving empty vector in the second surgery continued to lose
weight, although at a lower rate, and eventually the weight became
stable (FIG. 5f).
[0248] Flox-BDNF-expressing mice receiving Cre virus reversed the
progressive weight loss and commenced to regain weight gradually,
although their weight remained substantially lower than the
YFP-expressing obese mice (FIG. 5f).
[0249] At the end of the study, approximately 4 months after the
first surgery, both groups of BDNF-expressing mice showed markedly
lower body mass index than the YFP-expressing controls (FIG.
5g).
[0250] Because body weight influences bone density and is
considered as a risk factor for fracture, we measured the bone
mineral density of BDNF-expressing mice after considerable weight
loss and found no difference in either the whole-body skeleton
(excluding skull) or femur only (FIG. 5h), indicating a lack of
adverse effect on bones after BDNF-induced weight loss.
[0251] Discussion of Example I
[0252] Clinical gene transfer should ideally include some
regulatory control of therapeutic gene expression, particularly
when constitutive expression of the transgene may be deleterious.
Several pharmacological gene regulation technologies have been
developed. The Tet regulatory system, based on the use of small
molecules such as tetracycline or doxycycline, is the most widely
used. However, problems include the basal leakiness of the system
and the potential immunogenicity of the foreign proteins, in
addition to the need to administer a pharmacological agent with its
own attendant risks. A dimerizer-regulated approach such as the
rapamycin-FK506-binding protein system allows tight control in vivo
and is less likely to be immunogenic, because the key components of
this system are derived from human proteins. However, the size
limitation of the cloned sequences in rAAV requires splitting the
regulatory system into two separate vectors. The use of two
separate vectors is inefficient, owing to the need for double
infection of the host cell. In addition, it remains a question
whether the inducer drugs will be appropriate for the clinic.
[0253] Therefore, we constructed an autoregulatory system to
control therapeutic gene expression, mimicking the body's natural
molecular genetic feedback systems.
[0254] Here we show the efficacy of such an approach using BDNF as
the therapeutic gene, with weight loss and fat depletion as the
physiological readout and an AGRP promoter-driven microRNA cassette
as the regulatory agent. All of the components of this system can
be packaged into a single rAAV vector for efficient delivery. To
evaluate the regulation efficacy in vivo, we used a very high dose
of the vector in db/db mice. The unchecked overexpression of BDNF
led to marked weight loss, decrease of adiposity and improvement in
serum metabolic parameters. However, the weight of mice receiving
BDNF coupled with scrambled microRNA continued to drop (over 45% by
3 weeks after AAV injection) and showed no sign of stabilization,
ultimately requiring us to euthanize the mice. Moreover, the
BDNF-treated mice with severe weight loss and fat depletion,
including the wild-type mice on NCD and the db/db mice, both
treated with the nonregulated BDNF vectors, had a hyperactive
phenotype, and their immunocompetence was compromised (data not
shown). In contrast, the body weight of mice receiving an identical
dose of the autoregulatory vector, in which the constitutive BDNF
cassette was coupled with a physiologically responsive inhibitory
microRNA construct, had a more gradual weight loss without any
behavioral hyperactivity. The weight loss of these mice reached
.about.20% by 3 weeks after injection and then plateaued throughout
the entire 11-week duration of the experiment.
[0255] It is possible that by using experimental mice housed and
fed in a tightly controlled laboratory environment, we can titrate
the dose of vector to lead to the desirable weight loss without
overshoot. However, in a clinical setting, diet and activity may
vary unpredictably and from day to day. The efficacy of all current
antiobesity approaches is compromised by an inability to mandate
both a controlled lifestyle and energy-related activities, as well
as by noncompliance to any given diet. Moreover, although repeat
dosing is possible in gene therapy, every neurosurgical procedure
carries with it some risk, and one of the major advantages of
rAAV-mediated gene therapy is the potential for long-term, if not
permanent, treatment of a disorder after a single intervention.
[0256] The present invention enables a single dosing to which every
obese individual could respond and yet adapts and autoregulates
regardless of an individual's diet and lifestyle. The marked
alleviation of obesity that we observed was associated with loss of
liver steatosis, improvement in insulin sensitivity and glucose
tolerance, and reversal of hyperleptinemia and lipid
dyslipidemia.
[0257] In another aspect, the invention can be also used with other
molecular intervention studies in which expression of any given
functional transgene is self-regulated by a microRNA driven by
promoters activated, in turn, by the physiological changes induced
by the transgene of interest.
[0258] These data, in two obesity and diabetes models, show the
potency and long-term efficacy of hypothalamic gene transfer of
BDNF. Both suppression of food intake and heightening of energy
expenditure contribute to the weight loss of db/db mice receiving
BDNF, although the increase in energy expenditure of both basal
metabolism and spontaneous activity seems more noteworthy, given
that BDNF-overexpressing wild-type mice fed with either NCD or HFD
(while weight remained normal) did not change food intake.
[0259] Our data, though, are consistent with previous reports that
have shown that BDNF has a more potent effect on appetite in obese
mice. For example, chronic administration of BDNF protein
substantially suppressed food intake in mice with DIO but not those
fed on a standard diet. In addition, acute or chronic
administration of BDNF protein led to a marked decrease in food
intake in genetic obesity models, including yellow agouti mice and
db/db mice. This selective effect on appetite suppression in obese
mice is believed by the inventors herein to be explained by the
basal pattern of anorexic and/or orexigenic signaling in the
hypothalamus before BDNF treatment. The orexigenic Agrp and Npy
mRNA levels were approximately sixfold and threefold higher in
db/db mice than in wild-type mice, respectively, whereas the level
of anorexic Pomc was 30% lower (FIG. 9b).
[0260] The hyperphagia associated with these obesity models
(genetic or diet-induced obesity) reveals the appetite-suppressing
effect of BDNF gene therapy that is not apparent in euphagic mice.
We also showed that genes encoding proteins involved in energy
expenditure, such as Ucps, were considerably upregulated in the
liver and WAT of BDNF-expressing mice. Moreover, the comprehensive
analysis of gene expression in hypothalamus, liver and fat provides
further insights into the potential mechanisms underlying the
hypothalamic BDNF regulation of energy balance. For example
hypothalamic Crh expression was consistently increased in all of
the models we used and, accompanied by the increase in physical
activity, showing a potential pathway of BDNF regulation of the
hypothalamus-pituitary-adrenal axis, food intake and energy
expenditure.
[0261] In addition, WAT seems to be a primary peripheral organ
responsive to hypothalamic BDNF, with not only the display of much
smaller adipocytes but also substantial differences in the
expression profile of adipokines and genes involved in lipid
metabolism and mitochondrion activity. The WAT reduction was
primarily due to a reduction in cell size without much impact on
cellular viability, as shown by lack of adipocyte apoptosis (data
not shown) and reversible weight gain when the BDNF transgene was
knocked out by expression of Cre.
[0262] Thus, described herein are several strategies to achieve
potent and safe gene therapy for obesity and related metabolic
syndromes with AAV-BDNF vectors, including dose adjustment, an
autoregulatory negative feedback system using RNAi coupled to
transgene-induced physiological changes and, finally, a definitive
knockout via delivery of a second, rescue vector.
[0263] Long-term observation of mice receiving these therapeutic
vectors in both DIO and diabetic genetic models showed improved
general health, metabolic parameters and physical activity with no
adverse impact on bone density or disturbance in circadian rhythm
or home cage activity. The combination of these strategies will
further strengthen the safety of this gene therapy approach and
provide potent therapeutics for morbid obesity.
Example II
Vectors for Delivery of the Heterologous Protein
[0264] We developed vectors containing two cassettes, one cassette
expresses BDNF driven by a constitutive promoter CBA, the other
cassette expresses miR-BDNF driven by AGRP484.
[0265] Both transgene and the microRNA inhibiting the same
transgene can thus be delivered by a single virus. The constitutive
promoter CBA is much stronger than AGRP484. Therefore, the
transgene BDNF can achieve high level expression in hypothalamus
and lead to weight loss and associated physiological changes. Also,
in certain embodiments, a weaker promoter including cellular
promoters could also be used to drive the BDNF, since in the obese
state the AGRP promoter is dialed right down, but is activated when
weight is lost, so at target weight the AGRP promoter could be
stronger than the promoter driving the BDNF.
[0266] These strong changes can activate AGRP484 to express more
microRNA and subsequently tamper down the very high level of BDNF
and provide a negative feedback like regulation of transgene
expression responsive to physiological changes.
[0267] AAV vectors were injected into 3 groups of db/db mice which
were leptin receptor defective, obese and diabetic. Mice when
receiving YFP control virus continued to gain weight while
receiving BDNF plus scramble microRNA lost weight dramatically and
their weight continued to drop till sacrifice. On the contrary,
mice when given the BDNF plus AGRP-miR-BDNF, lost weight
significantly but stabilized between 3 to 4 weeks after
injection.
[0268] The vectors can be any vector suitable for delivering the
nucleic acid encoding a heterologous protein to a host cell at the
target site. The term "vector" as used herein refers to any genetic
element, such as a plasmid, phage, transposon, cosmid, chromosome,
artificial chromosome, virus, virion, non-viral vectors including
polymers, liposomes and various non-viral chemical complexes, and
the like. Thus, the term includes cloning and expression vehicles,
as well as viral vectors.
[0269] In one embodiment, the invention uses adeno-associated viral
vectors. AAV vectors can be constructed using known techniques to
provide at least the operatively linked components of control
elements including a transcriptional initiation region, a exogenous
nucleic acid molecule, a transcriptional termination region and at
least one post-transcriptional regulatory sequence. The control
elements are selected to be functional in the targeted cell. The
resulting construct which contains the operatively linked
components is flanked at the 5' and 3' region with functional AAV
ITR sequences.
[0270] The nucleotide sequences of AAV ITR regions are known. The
AAV ITRs are regions found at each end of the AAV genome which
function together in cis as origins of DNA replication and as
packaging signals for the viral genome. AAV ITRs, together with the
AAV rep coding region, provide for the efficient excision and
rescue from, and integration of a nucleotide sequence interposed
between two flanking ITRs into a mammalian cell genome. The ITR
sequences for AAV-2 are described, for example by Kotin et al.
(1994) Human Gene Therapy 5:793-801; Berns "Parvoviridae and their
Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields
and D. M. Knipe, eds.) The AAV-2 ITR have 145 nucleotides. The
terminal 125 nucleotides of each ITR form palindromic hairpin (HP)
structures that serve as primers for AAV DNA replication. Each ITR
also contains a stretch of 20 nucleotides, designated the D
sequence, which is not involved in hairpin structure formation.
(See e.g., Wang et al. (1998) J. Virol. 72: 5472-5480 and Wang et
al. (1997) J. Virol. 71: 3077-3082). Regions of the inverted
terminal repeats (ITR) are designated as A, B, C, A' and D at the
5'-end of the sequences and as D, A', B/C, C/B and A at the 3'-end
of the sequences. The site between these regions is referred to as
the terminal resolution site, which serves as a cleavage site in
the ITRs. For example, the Rep 78 and Rep 68 possess a number of
biochemical activities which include binding the viral inverted
terminal repeats (ITRs), nicking at the terminal resolution site,
and helicase activity. (See e.g., Kotin (1994) Hum. Gene Therap.
5:793-801 and Muzycza et al. (1992) 158: 97-129).
[0271] The skilled artisan will appreciate that AAV ITR's can be
modified using standard molecular biology techniques. Accordingly,
AAV ITR's used in the vectors of the invention need not have a
wild-type nucleotide sequence, and may be altered, e.g., by the
insertion, deletion or substitution of nucleotides. Additionally,
AAV ITR's may be derived from any of several AAV serotypes,
including but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,
AAV-6, AAVX7, and the like. Furthermore, 5' and 3' ITR's which
flank a selected nucleotide sequence in an AAV expression vector
need not necessarily be identical or derived from the same AAV
serotype or isolate, so long as the ITR's function as intended,
i.e., to allow for excision and replication of the bounded
nucleotide sequence of interest when AAV rep gene products are
present in the cell.
[0272] It is to be understood that, in certain embodiments, the AAV
vector genome can be single stranded containing the ITRs which
flank the genome; and in other embodiments, can be double stranded
so-called "self-complementary" (sc)AAV which also have ITRs
flanking the genome by one ITR which is altered. In one embodiment,
there is a deletion in the D-region of one of the ITRs which
prevents rep-mediated nicking of the newly synthesized rAAV genome
enabling efficient production and packaging of dimeric,
double-stranded rAAV genomes into recombinant sc particles.
[0273] The AAV rep coding region refers to a region of the AAV
genome which encodes the replication proteins of the virus which
are required to replicate the viral genome and to insert the viral
genome into a host genome during latent infection (Muzyczka, (1992)
Current Topics in Microbiol. and Immunol.; Bems, "Parvoviridae and
their Replication" in Fundamental Virology, 2d ed., (B. N. Fields
and D. M. Knipe, eds.). The term also includes functional
homologues thereof such as the human herpesvirus 6 (HHV-6) rep gene
which is also known to mediate AAV-2 DNA replication. The rep
coding region, as used herein, can be derived from any viral
serotype. The region need not include all of the wild-type genes
but may be altered, e.g., by the insertion, deletion or
substitution of nucleotides, so long as the rep genes function as
intended.
[0274] The AAV cap coding region refers to a region of the AAV
genome which encodes the coat proteins of the virus which are
required for packaging the viral genome. The AAV cap coding region,
as used herein, can be derived from any AAV serotype. The region
need not include all of the wild-type cap genes but may be altered,
e.g., by the insertion, deletion or substitution of nucleotides, so
long as the genes provide for sufficient packaging functions when
present in a host cell along with an AAV vector.
[0275] The AAV vectors can be derived from one or more
adeno-associated viruses, including without limitation, AAV-1,
AAV-2, AAV-3, AAV-4, AAV-5, AAV76, AAV-7, AAV-8, AVV-9, AVV-10. It
is to be understood that, in certain embodiments, the AAV vector(s)
can include AAV clades isolated from human and non-human primates
including recombinants or combinations and chimerics of such.
[0276] The AAV vectors can have one or more of the AAV wild-type
genes deleted in whole or part, preferably the rep and/or cap
genes, but retain functional flanking ITR sequences. Functional ITR
sequences are necessary for the rescue, replication and packaging
of the AAV virion. Thus, an AAV vector is defined herein to include
at least those sequences required in cis for replication and
packaging (e.g., functional ITRs) of the virus. The ITRs need not
be the wild-type nucleotide sequences, and may be altered, e.g., by
the insertion, deletion or substitution of nucleotides, so long as
the sequences provide for functional rescue, replication and
packaging.
[0277] The vectors can be produced using "AAV helper functions" or
"helpers" which refer to AAV-derived coding sequences that can be
expressed to provide AAV gene products that, in turn, function in
trans for productive AAV replication. Thus, AAV helper functions
include the rep and cap regions. The rep expression products have
been shown to possess many functions, including, among others:
recognition, binding and nicking of the AAV origin of DNA
replication; DNA helicase activity; and modulation of transcription
from AAV (or other heterologous) promoters. The cap expression
products supply necessary packaging functions. AAV helper functions
are used herein to complement AAV functions in trans that are
missing from AAV vectors.
[0278] An AAV helper construct refers generally to a nucleic acid
molecule that includes nucleotide sequences which provide AAV
functions. These AAV functions include the rep and cap coding
regions that are replaced by a nucleotide sequence of interest in
an AAV delivery vector. AAV helper constructs are commonly used to
provide transient expression of AAV rep and/or cap genes to
complement missing AAV functions that are necessary for lytic AAV
replication; however, all previous helper constructs lack AAV ITRs
and can neither replicate nor package themselves. AAV helper
constructs can be in the form of a plasmid, phage, transposon,
cosmid, virus, or virion. A number of other vectors have been
described which encode Rep and/or Cap expression products. See,
e.g., U.S. Pat. No. 5,139,941. The helper constructs of present
invention include at least one copy of AAV ITR or functional
equivalent to make it competent for AAV replication and rescue.
[0279] It may also be necessary to provide "accessory functions"
which refer to non-AAV derived viral and/or cellular functions upon
which AAV is dependent for its replication (Carter, (1990)
"Adeno-Associated Virus Helper Functions," in CRC Handbook of
Parvoviruses, vol. I (P. Tijssen, ed.)). Thus, the term captures
DNAs, RNAs and protein that are required for AAV replication,
including those moieties involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of Cap expression products and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1) and vaccinia virus.
[0280] Accessory functions can be provided by "accessory function
vector" which refer generally to a nucleic acid molecule that
includes nucleotide sequences providing accessory functions. An
accessory function vector can be transfected into a suitable host
cell, wherein the vector is then capable of supporting AAV virion
production in the host cell. Expressly excluded from the term are
infectious viral particles as they exist in nature, such as
adenovirus, herpesvirus or vaccinia virus particles. Thus,
accessory function vectors can be in the form of a plasmid, phage,
transposon, cosmid or virus that has been modified from its
naturally occurring form.
[0281] The skilled artisan can appreciate that regulatory sequences
can often be provided from commonly used promoters derived from
viruses such as, polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40. Use of viral regulatory elements to direct expression of
the protein can allow for high level constitutive expression of the
protein in a variety of host cells. Ubiquitously expressing
promoters can also be used include, for example, the early
cytomegalovirus promoter Boshart et al. (1985) Cell 41:521-530,
herpesvirus thymidine kinase (HSV-TK) promoter (McKnight et al.
(1984) Cell 37: 253-262), beta-actin promoters (e.g., the human
.beta.-actin promoter as described by Ng et al. (1985) Mol. Cell
Biol. 5: 2720-2732) and colony stimulating factor-1 (CSF-1)
promoter (Ladner et al. (1987) EMBO J. 6: 2693-2698).
[0282] Alternatively, the regulatory sequences of the AAV vector
can direct expression of the transgene preferentially in a
particular cell type, i.e., tissue-specific regulatory elements can
be used. Non-limiting examples of tissue-specific promoters which
can be used include, central nervous system (CNS) specific
promoters such as, neuron-specific promoters (e.g., the
neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad.
Sci. USA 86:5473-5477) and glial specific promoters (Morii et al.
(1991) Biochem. Biophys Res. Commun. 175: 185-191).
[0283] The AAV vector harboring the transgene flanked by AAV ITRs,
can be constructed by directly inserting the transgene into an AAV
genome which has had the major AAV open reading frames ("ORFs")
excised therefrom. Other portions of the AAV genome can also be
deleted, as long as a sufficient portion of the ITRs remain to
allow for replication and packaging functions. These constructs can
be designed using techniques well known in the art. (See, e.g.,
Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et
al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);
Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka
(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin
(1994) Human Gene Therapy 5:793-801; Shelling et al. (1994) Gene
Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med.
179:1867-1875).
[0284] Several AAV vectors are available from the American Type
Culture Collection ("ATCC") under Accession Numbers 53222, 53223,
53224, 53225 and 53226.
[0285] The AAV vectors can be transfected into a host cell with a
helper function, e.g., a helper function plasmid (See Section II)
and/or accessory functions to produce recombinant AAV virions
(rAAV). Recombinant AAV virions (rAAV) refer to an infectious,
replication-defective virus composed of an AAV protein shell
encapsidating a nucleotide sequence encoding a therapeutic protein
that is flanked on both sides by AAV ITRs. A number of transfection
techniques are generally known in the art. See, e.g., Graham et al.
(1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning,
a laboratory manual, Cold Spring Harbor Laboratories, N.Y., Davis
et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu
et al. (1981) Gene 13:197. Particularly suitable transfection
methods include calcium phosphate co-precipitation (Graham et al.
(1973) Virol. 52:456-467), direct micro-injection into cultured
cells (Capecchi (1980) Cell 22:479-488), electroporation (Shigekawa
et al. (1988) BioTechniques 6:742-751), liposome mediated gene
transfer (Mannino et al. (1988) BioTechniques 6:682-690),
lipid-mediated transduction (Felgner et al. (1987) Proc. Natl.
Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using
high-velocity microprojectiles (Klein et al. (1987) Nature
327:70-73).
[0286] Suitable host cells for producing recombinant AAV virions
include, but are not limited to, microorganisms, yeast cells,
insect cells, and mammalian cells, that can be, or have been, used
as recipients of a exogenous nucleic acid molecule. Thus, a "host
cell" as used herein generally refers to a cell which has been
transfected with an exogenous nucleic acid molecule. The host cell
includes any eukaryotic cell or cell line so long as the cell or
cell line is not incompatible with the protein to be expressed, the
selection system chosen or the fermentation system employed.
[0287] In one embodiment, cells from the stable human cell line,
293 (readily available through, e.g., the ATCC under Accession No.
ATCC CRL1573) are especially useful. Particularly, the human cell
line 293, which is a human embryonic kidney cell line that has been
transformed with adenovirus type-5 DNA fragments (Graham et al.
(1977) J. Gen. Virol. 36:59), and expresses the adenoviral EIA and
E1B genes (Aiello et al. (1979) Virology 94:460). The 293 cell line
is readily transfected, and provides a particularly convenient
platform in which to produce recombinant AAV virions.
[0288] For example, FIG. 13a shows the pAM/CBA-NPY-WPRE-BGH plasmid
map, and FIG. 13b shows the pAM/CBA-NPY-WPRE-BGH nucleotide
sequence [SEQ ID NO:1]. FIG. 13c shows the CAG-BDNF-HA-WPRE
sequence [SEQ ID NO:2].
[0289] FIG. 14 shows two targeting sequences with the highest
scores (Invitrogene RNAi Design Tool) were selected and cloned into
the Block-iT PolII miR RNAi expression vector: WPRE 74:
CTATGTGGACGCTGCTTTA [SEQ ID NO:3], and WPRE155: TCCTGGTTTGTCTCTTTAT
[SEQ ID NO:4]. In in vitro experiments, both miR constructs
inhibited BDNF expression by at least 90% when co-transfected with
the HA-BDNF-WPRE plasmid, as confirmed by ELISA for BDNF.
miR-WPRE74 was chosen to construct the autoregulatory plasmid
shown.
[0290] FIG. 15: The mRNA for human BDNF [SEQ ID NO:5].
[0291] FIG. 16: The mRNA for human trkB [SEQ ID NO:6].
[0292] FIG. 17: DNA sequence and gene structure of human AGRP [SEQ
ID NO:7].
[0293] FIG. 18: DNA sequence for woodchuck post-transcriptional
regulatory element (WPRE) [SEQ ID NO:8].
Example III
Delivery of the Heterologous Protein to the Target Site
[0294] The vectors carrying the nucleic acid encoding at least one
heterologous protein can be precisely delivered into specific sites
of the central nervous system, and in particular the brain, using
stereotactic microinjection techniques. For example, the subject
being treated can be placed within a stereotactic frame base
(MRI-compatible) and then imaged using high resolution MRI to
determine the three-dimensional positioning of the particular
region to be treated. The MRI images can then be transferred to a
computer having the appropriate stereotactic software, and a number
of images are used to determine a target site and trajectory for
pharmacological agent microinjection. The software translates the
trajectory into three-dimensional coordinates that are precisely
registered for the stereotactic frame. In the case of intracranial
delivery, the skull will be exposed, burr holes will be drilled
above the entry site, and the stereotactic apparatus used to
position the needle and ensure implantation at a predetermined
depth. The pharmacological agent can be delivered to regions, such
as the cells of the spinal cord, brainstem, or brain that are
associated with the disease or disorder. For example, target
regions can include the medulla, pons, and midbrain, cerebellum,
diencephalons (e.g., thalamus, hypothalamus), telencephalon (e.g.,
corpus stratium, cerebral cortex, or within the cortex, the
occipital, temporal, parietal or frontal lobes), or combinations,
thereof.
[0295] One particular route of delivery is an approach via the
ventricles, with or without an endoscope, typically via the lateral
ventricle, through to the third ventricle which lies immediately
adjacent to the hypothalamus. Another approach is transnasally,
typically transphenoidally, in which a direct approach through the
nasal sinuses to the base of the brain, and then a device inserted
to deliver the vector directly through this skull base approach
into the hypothalamus. Still another approach can be to deliver the
vector simply into the ventricles with sufficient hypothalamic
expression obtained to induce weight loss.
Example IV
Delivery of Therapeutic Agents, Pharmaceutical Compositions and
Pharmaceutical Administration
[0296] The therapeutic agents of the invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises the
vector of the invention and a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Examples of
pharmaceutically acceptable carriers include one or more of water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. In many cases, it will
be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Pharmaceutically acceptable carriers may further
comprise minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the
shelf life or effectiveness of the antibody or antibody
portion.
[0297] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans. The preferred mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In one embodiment, the vector is
administered by intravenous infusion or injection. In another
embodiment, the vector is administered by intramuscular, by
subcutaneous injection, or perorally.
[0298] In the most preferred embodiment, the vector is delivered to
a specific location using stereotactic delivery.
[0299] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antigen, antibody or
antibody portion) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization.
[0300] Generally, dispersions are prepared by incorporating the
active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile, lyophilized powders for
the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum drying and spray-drying that
yields a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof. The proper fluidity of a solution can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prolonged absorption of injectable
compositions can be brought about by including in the composition
an agent that delays absorption, for example, monostearate salts
and gelatin.
[0301] The vector of the present invention can be administered by a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0302] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of the vectors of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the vector may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the
vector to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the vector are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result.
Typically, since a prophylactic dose is used in subjects prior to
or at an earlier stage of disease, the prophylactically effective
amount will be less than the therapeutically effective amount.
[0303] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0304] One skilled in the art will appreciate further features and
advantages of the invention based on the above-described
embodiments. Accordingly, the invention is not to be limited by
what has been particularly shown and described, except as indicated
by the appended claims. All publications and references cited
herein are expressly incorporated herein by reference in their
entirety.
[0305] While the invention has been described with reference to
various and preferred embodiments, it should be understood by those
skilled in the art that various changes may be made and equivalents
may be substituted for elements thereof without departing from the
essential scope of the invention. In addition, many modifications
may be made to adapt a particular situation or material to the
teachings of the invention without departing from the essential
scope thereof. Therefore, it is intended that the invention not be
limited to the particular embodiment disclosed herein contemplated
for carrying out this invention, but that the invention will
include all embodiments falling within the scope of the claims.
Sequence CWU 1
1
1215641DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1tagctgcgcg ctcgctcgct cactgaggcc
gcccgggcaa agcccgggcg tcgggcgacc 60tttggtcgcc cggcctcagt gagcgagcga
gcgcgcagag agggagtggc caactccatc 120actaggggtt ccttgtagtt
aatgattaac ccgccatgct acttatctac gtagccatgc 180tctaggtacc
attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
240tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
gtacatcaag 300tgtatcatat gccaagtacg ccccctattg acgtcaatga
cggtaaatgg cccgcctggc 360attatgccca gtacatgacc ttatgggact
ttcctacttg gcagtacatc tacgtattag 420tcatcgctat taccatggtc
gaggtgagcc ccacgttctg cttcactctc cccatctccc 480ccccctcccc
acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
540gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag
gggcggggcg 600gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
cgcgctccga aagtttcctt 660ttatggcgag gcggcggcgg cggcggccct
ataaaaagcg aagcgcgcgg cgggcgggag 720tcgctgcgac gctgccttcg
ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc 780cggctctgac
tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
840ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct
gcgtgaaagc 900cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
gctcggggct gtccgcgggg 960ggacggctgc cttcgggggg gacggggcag
ggcggggttc ggcttctggc gtgtgaccgg 1020cggctctaga gcctctgcta
accatgttca tgccttcttc tttttcctac agctcctggg 1080caacgtgctg
gttattgtgc tgtctcatca ttttggcaaa gaattggatc cgccatgcta
1140ggtaacaagc gactggggct gtccggactg accctcgccc tgtccctgct
cgtgtgcctg 1200ggtgcgctgg ccgaggcgta cccctccaag ccggacaacc
cgggcgagga cgcaccagcg 1260gaggacatgg ccagatacta ctcagcgctg
cgacactaca tcaacctcat caccaggcag 1320agatatggaa aacgatctag
cccagagaca ctgatttcag acctcttgat gagagaaagc 1380acagaaaatg
ttcccagaac tcggcttgaa gaccctgcaa tgtggtgaga attcgatatc
1440aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
tggtattctt 1500aactatgttg ctccttttac gctatgtgga tacgctgctt
taatgccttt gtatcatgct 1560attgcttccc gtatggcttt cattttctcc
tccttgtata aatcctggtt gctgtctctt 1620tatgaggagt tgtggcccgt
tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 1680gcaaccccca
ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
1740ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
ctgctggaca 1800ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
cggggaaatc atcgtccttt 1860ccttggctgc tcgcctgtgt tgccacctgg
attctgcgcg ggacgtcctt ctgctacgtc 1920ccttcggccc tcaatccagc
ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 1980cttccgcgtc
ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
2040catcgatacc gtcgactcgc tgatcagcct cgactgtgcc ttctagttgc
cagccatctg 2100ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
tgccactccc actgtccttt 2160cctaataaaa tgaggaaatt gcatcgcatt
gtctgagtag gtgtcattct attctggggg 2220gtggggtggg gcaggacagc
aagggggagg attgggaaga caatagcagg catgctgggg 2280atgcggtggg
ctctatggct tctgaggcgg aaagaaccag ctggggctcg actagagcat
2340ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc
cctagtgatg 2400gagttggcca ctccctctct gcgcgctcgc tcgctcactg
aggccgggcg accaaaggtc 2460gcccgacgcc cgggctttgc ccgggcggcc
tcagtgagcg agcgagcgcg cagagctttt 2520tgcaaaagcc taggcctcca
aaaaagcctc ctcactactt ctggaatagc tcagaggccg 2580aggcggcctc
ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
2640cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
atggttgctg 2700actaattgag atgcatgctt tgcatacttc tgcctgctgg
ggagcctggg gactttccac 2760acctggttgc tgactaattg agatgcatgc
tttgcatact tctgcctgct ggggagcctg 2820gggactttcc acaccctaac
tgacacacat tccacagctg cattaatgaa tcggccaacg 2880cgcggggaga
ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
2940gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
taatacggtt 3000atccacagaa tcaggggata acgcaggaaa gaacatgtga
gcaaaaggcc agcaaaaggc 3060caggaaccgt aaaaaggccg cgttgctggc
gtttttccat aggctccgcc cccctgacga 3120gcatcacaaa aatcgacgct
caagtcagag gtggcgaaac ccgacaggac tataaagata 3180ccaggcgttt
ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
3240cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
gctcacgctg 3300taggtatctc agttcggtgt aggtcgttcg ctccaagctg
ggctgtgtgc acgaaccccc 3360cgttcagccc gaccgctgcg ccttatccgg
taactatcgt cttgagtcca acccggtaag 3420acacgactta tcgccactgg
cagcagccac tggtaacagg attagcagag cgaggtatgt 3480aggcggtgct
acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
3540atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
gtagctcttg 3600atccggcaaa caaaccaccg ctggtagcgg tggttttttt
gtttgcaagc agcagattac 3660gcgcagaaaa aaaggatctc aagaagatcc
tttgatcttt tctacggggt ctgacgctca 3720gtggaacgaa aactcacgtt
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 3780ctagatcctt
ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
3840ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
tctgtctatt 3900tcgttcatcc atagttgcct gactccccgt cgtgtagata
actacgatac gggagggctt 3960accatctggc cccagtgctg caatgatacc
gcgagaccca cgctcaccgg ctccagattt 4020atcagcaata aaccagccag
ccggaagggc cgagcgcaga agtggtcctg caactttatc 4080cgcctccatc
cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
4140tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
cgtcgtttgg 4200tatggcttca ttcagctccg gttcccaacg atcaaggcga
gttacatgat cccccatgtt 4260gtgcaaaaaa gcggttagct ccttcggtcc
tccgatcgtt gtcagaagta agttggccgc 4320agtgttatca ctcatggtta
tggcagcact gcataattct cttactgtca tgccatccgt 4380aagatgcttt
tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
4440gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
atagcagaac 4500tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
aaactctcaa ggatcttacc 4560gctgttgaga tccagttcga tgtaacccac
tcgtgcaccc aactgatctt cagcatcttt 4620tactttcacc agcgtttctg
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 4680aataagggcg
acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
4740catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
agaaaaataa 4800acaaataggg gttccgcgca catttccccg aaaagtgcca
cctgacgtct aagaaaccat 4860tattatcatg acattaacct ataaaaatag
gcgtatcacg aggccctttc gtctcgcgcg 4920tttcggtgat gacggtgaaa
acctctgaca catgcagctc ccggagacgg tcacagcttg 4980tctgtaagcg
gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
5040gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
tgcaccattc 5100gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
cagtagtagg ttgaggccgt 5160tgagcaccgc cgccgcaagg aatggtgcat
gcaaggagat ggcgcccaac agtcccccgg 5220ccacggggcc tgccaccata
cccacgccga aacaagcgct catgagcccg aagtggcgag 5280cccgatcttc
cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
5340cggtgatgcc ggccacgatg cgtccggcgt agaggatctg gctagcgatg
accctgctga 5400ttggttcgct gaccatttcc gggtgcggga cggcgttacc
agaaactcag aaggttcgtc 5460caaccaaacc gactctgacg gcagtttacg
agagagatga tagggtctgc ttcagtaagc 5520cagatgctac acaattaggc
ttgtacatat tgtcgttaga acgcggctac aattaataca 5580taaccttatg
tatcatacac atacgattta ggtgacacta tagaatacac ggaattaatt 5640c
564122365DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 2cattgacgtc aataatgacg tatgttccca
tagtaacgcc aatagggact ttccattgac 60gtcaatgggt ggactattta cggtaaactg
cccacttggc agtacatcaa gtgtatcata 120tgccaagtac gccccctatt
gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 180agtacatgac
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
240ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
cccccctccc 300cacccccaat tttgtattta tttatttttt aattattttg
tgcagcgatg ggggcggggg 360gggggggggg gcgcgcgcca ggcggggcgg
ggcggggcga ggggcggggc ggggcgaggc 420ggagaggtgc ggcggcagcc
aatcagagcg gcgcgctccg aaagtttcct tttatggcga 480ggcggcggcg
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
540cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
ccggctctga 600ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
cttctcctcc gggctgtaat 660tagcgcttgg tttaatgacg gcttgtttct
tttctgtggc tgcgtgaaag ccttgagggg 720ctccgggagg gccctttgtg
cggggggagc ggctcggggc tgtccgcggg gggacggctg 780ccttcggggg
ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
840agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
gcaacgtgct 900ggttattgtg ctgtctcatc attttggcaa agaattggat
ccactcgagt ggagctcgcg 960actagtcgat tcgaattcgg cttgtgatga
ccatcctttt ccttactatg gttatttcat 1020actttggttg catgaaggct
gcccccatga aagaagcaaa catccgagga caaggtggct 1080tggcctaccc
aggtgtgcgg acccatggga ctctggagag cgtgaatggg cccaaggcag
1140gttcaagagg cttgacatca ttggctgaca ctttcgaaca cgtgatagaa
gagctgttgg 1200atgaggacca gaaagttcgg cccaatgaag aaaacaataa
ggacgcagac ttgtacacgt 1260ccagggtgat gctcagtagt caagtgcctt
tggagcctcc tcttctcttt ctgctggagg 1320aatacaaaaa ttacctagat
gctgcaaaca tgtccatgag ggtccggcgc cactctgacc 1380ctgcccgccg
aggggagctg agcgtgtgtg acagtattag tgagtgggta acggcggcag
1440acaaaaagac tgcagtggac atgtcgggcg ggacggtcac agtccttgaa
aaggtccctg 1500tatcaaaagg ccaactgaag caatacttct acgagaccaa
gtgcaatccc atgggttaca 1560caaaagaagg ctgcaggggc atagacaaaa
ggcattggaa ctcccagtgc cgaactaccc 1620agtcgtacgt gcgggccctt
accatggata gcaaaaagag aattggctgg cgattcataa 1680ggatagacac
ttcttgtgta tgtacattga ccattaaaag gggaagatat ccgtatgatg
1740ttcctgatta ttgagaattc gatatcaagc ttatcgataa tcaacctctg
gattacaaaa 1800tttgtgaaag attgactggt attcttaact atgttgctcc
ttttacgcta tgtggatacg 1860ctgctttaat gcctttgtat catgctattg
cttcccgtat ggctttcatt ttctcctcct 1920tgtataaatc ctggttgctg
tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 1980gcgtggtgtg
cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
2040gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
gaactcatcg 2100ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
gggcactgac aattccgtgg 2160tgttgtcggg gaaatcatcg tcctttcctt
ggctgctcgc ctgtgttgcc acctggattc 2220tgcgcgggac gtccttctgc
tacgtccctt cggccctcaa tccagcggac cttccttccc 2280gcggcctgct
gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
2340ggatctccct ttgggccgcc tcccc 2365319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3ctatgtggac gctgcttta 19419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4tcctggtttg tctctttat 1954755DNAHomo sapiens
5cacacacaca cacacacaca gagagaacat ctctagtaaa aagaaaagtt gagctttctt
60agctagatgt gtgtattagc cagaaaaagc caaggagtga agggttttag agaactggag
120gagataaagt ggagtctgca tatgggaggc atttgaaatg gacttaaatg
tctttttaat 180gctgactttt tcagttttct ccttaccaga cacattgttt
tcatgacatt agccccaggc 240atagacacat cattaaaatg aacatgtcaa
aaaatgattt ctgtttagaa ataagcaaaa 300cattttcagt tgtgaccacc
caggtgtaga ataaagaaca gtggaattgg gagccctgag 360ttctaacata
aactttcttc atgacataag gcaagtcttc tatggccttt ggtttcctta
420cctgtaaaac aggatggctc aatgaaatta tctttcttct ttgctataat
agagtatctc 480tgtgggaaga ggaaaaaaaa agtcaattta aaggctcctt
atagttcccc aactgctgtt 540ttattgtgct attcatgcct agacatcaca
tagctagaaa ggcccatcag acccctcagg 600ccactgctgt tcctgtcaca
cattcctgca aaggaccatg ttgctaactt gaaaaaaatt 660actattaatt
acacttgcag ttgttgctta gtaacattta tgattttgtg tttctcgtga
720cagcatgagc agagatcatt aaaaattaaa cttacaaagc tgctaaagtg
ggaagaagga 780gaacttgaag ccacaatttt tgcacttgct tagaagccat
ctaatctcag gttatatgct 840agatcttggg ggcaaacact gcatgtctct
ggtttatatt aaaccacata cagcacacta 900ctgacactga tttgtgtctg
gtgcagctgg agtttatcac caagacataa aaaaaccttg 960accctgcaga
atggcctgga attacaatca gatgggccac atggcatccc ggtgaaagaa
1020agccctaacc agttttctgt cttgtttctg ctttctccct acagttccac
caggtgagaa 1080gagtgatgac catccttttc cttactatgg ttatttcata
ctttggttgc atgaaggctg 1140cccccatgaa agaagcaaac atccgaggac
aaggtggctt ggcctaccca ggtgtgcgga 1200cccatgggac tctggagagc
gtgaatgggc ccaaggcagg ttcaagaggc ttgacatcat 1260tggctgacac
tttcgaacac gtgatagaag agctgttgga tgaggaccag aaagttcggc
1320ccaatgaaga aaacaataag gacgcagact tgtacacgtc cagggtgatg
ctcagtagtc 1380aagtgccttt ggagcctcct cttctctttc tgctggagga
atacaaaaat tacctagatg 1440ctgcaaacat gtccatgagg gtccggcgcc
actctgaccc tgcccgccga ggggagctga 1500gcgtgtgtga cagtattagt
gagtgggtaa cggcggcaga caaaaagact gcagtggaca 1560tgtcgggcgg
gacggtcaca gtccttgaaa aggtccctgt atcaaaaggc caactgaagc
1620aatacttcta cgagaccaag tgcaatccca tgggttacac aaaagaaggc
tgcaggggca 1680tagacaaaag gcattggaac tcccagtgcc gaactaccca
gtcgtacgtg cgggccctta 1740ccatggatag caaaaagaga attggctggc
gattcataag gatagacact tcttgtgtat 1800gtacattgac cattaaaagg
ggaagatagt ggatttatgt tgtatagatt agattatatt 1860gagacaaaaa
ttatctattt gtatatatac ataacagggt aaattattca gttaagaaaa
1920aaataatttt atgaactgca tgtataaatg aagtttatac agtacagtgg
ttctacaatc 1980tatttattgg acatgtccat gaccagaagg gaaacagtca
tttgcgcaca acttaaaaag 2040tctgcattac attccttgat aatgttgtgg
tttgttgccg ttgccaagaa ctgaaaacat 2100aaaaagttaa aaaaaataat
aaattgcatg ctgctttaat tgtgaattga taataaactg 2160tcctctttca
gaaaacagaa aaaaacacac acacacacaa caaaaatttg aaccaaaaca
2220ttccgtttac attttagaca gtaagtatct tcgttcttgt tagtactata
tctgttttac 2280tgcttttaac ttctgatagc gttggaatta aaacaatgtc
aaggtgctgt tgtcattgct 2340ttactggctt aggggatggg ggatgggggg
tatatttttg tttgttttgt gttttttttt 2400cgtttgtttg ttttgttttt
tagttcccac agggagtaga gatggggaaa gaattcctac 2460aatatatatt
ctggctgata aaagatacat ttgtatgttg tgaagatgtt tgcaatatcg
2520atcagatgac tagaaagtga ataaaaatta aggcaactga acaaaaaaat
gctcacactc 2580cacatcccgt gatgcacctc ccaggccccg ctcattcttt
gggcgttggt cagagtaagc 2640tgcttttgac ggaaggacct atgtttgctc
agaacacatt ctttcccccc ctccccctct 2700ggtctcctct ttgttttgtt
ttaaggaaga aaaatcagtt gcgcgttctg aaatatttta 2760ccactgctgt
gaacaagtga acacattgtg tcacatcatg acactcgtat aagcatggag
2820aacagtgatt tttttttaga acagaaaaca acaaaaaata accccaaaat
gaagattatt 2880ttttatgagg agtgaacatt tgggtaaatc atggctaagc
ttaaaaaaaa ctcatggtga 2940ggcttaacaa tgtcttgtaa gcaaaaggta
gagccctgta tcaacccaga aacacctaga 3000tcagaacagg aatccacatt
gccagtgaca tgagactgaa cagccaaatg gaggctatgt 3060ggagttggca
ttgcatttac cggcagtgcg ggaggaattt ctgagtggcc atcccaaggt
3120ctaggtggag gtggggcatg gtatttgaga cattccaaaa cgaaggcctc
tgaaggaccc 3180ttcagaggtg gctctggaat gacatgtgtc aagctgcttg
gacctcgtgc tttaagtgcc 3240tacattatct aactgtgctc aagaggttct
cgactggagg accacactca agccgactta 3300tgcccaccat cccacctctg
gataattttg cataaaattg gattagcctg gagcaggttg 3360ggagccaaat
gtggcatttg tgatcatgag attgatgcaa tgagatagaa gatgtttgct
3420acctgaacac ttattgcttt gaaactagac ttgaggaaac cagggtttat
cttttgagaa 3480cttttggtaa gggaaaaggg aacaggaaaa gaaaccccaa
actcaggccg aatgatcaag 3540gggacccata ggaaatcttg tccagagaca
agacttcggg aaggtgtctg gacattcaga 3600acaccaagac ttgaaggtgc
cttgctcaat ggaagaggcc aggacagagc tgacaaaatt 3660ttgctcccca
gtgaaggcca cagcaacctt ctgcccatcc tgtctgttca tggagagggt
3720ccctgcctca cctctgccat tttgggttag gagaagtcaa gttgggagcc
tgaaatagtg 3780gttcttggaa aaatggatcc ccagtgaaaa ctagagctct
aagcccattc agcccatttc 3840acacctgaaa atgttagtga tcaccacttg
gaccagcatc cttaagtatc agaaagcccc 3900aagcaattgc tgcatcttag
tagggtgagg gataagcaaa agaggatgtt caccataacc 3960caggaatgaa
gataccatca gcaaagaatt tcaatttgtt cagtctttca tttagagcta
4020gtctttcaca gtaccatctg aatacctctt tgaaagaagg aagactttac
gtagtgtaga 4080tttgttttgt gttgtttgaa aatattatct ttgtaattat
ttttaatatg taaggaatgc 4140ttggaatatc tgctatatgt caactttatg
cagcttcctt ttgagggaca aatttaaaac 4200aaacaacccc ccatcacaaa
cttaaaggat tgcaagggcc agatctgtta agtggtttca 4260taggagacac
atccagcaat tgtgtggtca gtggctcttt tacccaataa gatacatcac
4320agtcacatgc ttgatggttt atgttgacct aagatttatt ttgttaaaat
ctctctctgt 4380tgtgttcgtt cttgttctgt tttgttttgt tttttaaagt
cttgctgtgg tctctttgtg 4440gcagaagtgt ttcatgcatg gcagcaggcc
tgttgctttt ttatggcgat tcccattgaa 4500aatgtaagta aatgtctgtg
gccttgttct ctctatggta aagatattat tcaccatgta 4560aaacaaaaaa
caatatttat tgtattttag tatatttata taattatgtt attgaaaaaa
4620attggcatta aaacttaacc gcatcagaac ctattgtaaa tacaagttct
atttaagtgt 4680actaattaac atataatata tgttttaaat atagaatttt
taatgttttt aaatatattt 4740tcaaagtaca taaaa 475565608DNAHomo sapiens
6aagacggatt ctcagacaag gcttgcaaat gccccgcagc catcatttaa ctgcacccgc
60agaatagtta cggtttgtca cccgaccctc ccggatcgcc taatttgtcc ctagtgagac
120cccgaggctc tgcccgcgcc tggcttcttc gtagctggat gcatatcgtg
ctccgggcag 180cgcgggcgca gggcacgcgt tcgcgcacac cctagcacac
atgaacacgc gcaagagctg 240aaccaagcac ggtttccatt tcaaaaaggg
agacagcctc taccgcgatt gtagaagaga 300ctgtggtgtg aattagggac
cgggaggcgt cgaacggagg aacggttcat cttagagact 360aattttctgg
agtttctgcc cctgctctgc gtcagccctc acgtcacttc gccagcagta
420gcagaggcgg cggcggcggc tcccggaatt gggttggagc aggagcctcg
ctggctgctt 480cgctcgcgct ctacgcgctc agtccccggc ggtagcagga
gcctggaccc aggcgccgcc 540ggcgggcgtg aggcgccgga gcccggcctc
gaggtgcata ccggaccccc attcgcatct 600aacaaggaat ctgcgcccca
gagagtcccg ggagcgccgc cggtcggtgc ccggcgcgcc 660gggccatgca
gcgacggccg ccgcggagct ccgagcagcg gtagcgcccc cctgtaaagc
720ggttcgctat gccggggcca ctgtgaaccc tgccgcctgc cggaacactc
ttcgctccgg 780accagctcag cctctgataa gctggactcg gcacgcccgc
aacaagcacc gaggagttaa 840gagagccgca agcgcaggga aggcctcccc
gcacgggtgg gggaaagcgg ccggtgcagc 900gcggggacag gcactcgggc
tggcactggc tgctagggat gtcgtcctgg ataaggtggc 960atggacccgc
catggcgcgg ctctggggct tctgctggct ggttgtgggc ttctggaggg
1020ccgctttcgc ctgtcccacg tcctgcaaat gcagtgcctc tcggatctgg
tgcagcgacc 1080cttctcctgg catcgtggca tttccgagat tggagcctaa
cagtgtagat cctgagaaca 1140tcaccgaaat tttcatcgca aaccagaaaa
ggttagaaat catcaacgaa gatgatgttg 1200aagcttatgt gggactgaga
aatctgacaa ttgtggattc tggattaaaa tttgtggctc 1260ataaagcatt
tctgaaaaac agcaacctgc agcacatcaa ttttacccga aacaaactga
1320cgagtttgtc taggaaacat ttccgtcacc ttgacttgtc tgaactgatc
ctggtgggca 1380atccatttac atgctcctgt gacattatgt ggatcaagac
tctccaagag gctaaatcca 1440gtccagacac tcaggatttg tactgcctga
atgaaagcag caagaatatt cccctggcaa 1500acctgcagat acccaattgt
ggtttgccat ctgcaaatct ggccgcacct aacctcactg 1560tggaggaagg
aaagtctatc acattatcct gtagtgtggc aggtgatccg gttcctaata
1620tgtattggga tgttggtaac ctggtttcca aacatatgaa tgaaacaagc
cacacacagg 1680gctccttaag
gataactaac atttcatccg atgacagtgg gaagcagatc tcttgtgtgg
1740cggaaaatct tgtaggagaa gatcaagatt ctgtcaacct cactgtgcat
tttgcaccaa 1800ctatcacatt tctcgaatct ccaacctcag accaccactg
gtgcattcca ttcactgtga 1860aaggcaaccc caaaccagcg cttcagtggt
tctataacgg ggcaatattg aatgagtcca 1920aatacatctg tactaaaata
catgttacca atcacacgga gtaccacggc tgcctccagc 1980tggataatcc
cactcacatg aacaatgggg actacactct aatagccaag aatgagtatg
2040ggaaggatga gaaacagatt tctgctcact tcatgggctg gcctggaatt
gacgatggtg 2100caaacccaaa ttatcctgat gtaatttatg aagattatgg
aactgcagcg aatgacatcg 2160gggacaccac gaacagaagt aatgaaatcc
cttccacaga cgtcactgat aaaaccggtc 2220gggaacatct ctcggtctat
gctgtggtgg tgattgcgtc tgtggtggga ttttgccttt 2280tggtaatgct
gtttctgctt aagttggcaa gacactccaa gtttggcatg aaagatttct
2340catggtttgg atttgggaaa gtaaaatcaa gacaaggtgt tggcccagcc
tccgttatca 2400gcaatgatga tgactctgcc agcccactcc atcacatctc
caatgggagt aacactccat 2460cttcttcgga aggtggccca gatgctgtca
ttattggaat gaccaagatc cctgtcattg 2520aaaatcccca gtactttggc
atcaccaaca gtcagctcaa gccagacaca tttgttcagc 2580acatcaagcg
acataacatt gttctgaaaa gggagctagg cgaaggagcc tttggaaaag
2640tgttcctagc tgaatgctat aacctctgtc ctgagcagga caagatcttg
gtggcagtga 2700agaccctgaa ggatgccagt gacaatgcac gcaaggactt
ccaccgtgag gccgagctcc 2760tgaccaacct ccagcatgag cacatcgtca
agttctatgg cgtctgcgtg gagggcgacc 2820ccctcatcat ggtctttgag
tacatgaagc atggggacct caacaagttc ctcagggcac 2880acggccctga
tgccgtgctg atggctgagg gcaacccgcc cacggaactg acgcagtcgc
2940agatgctgca tatagcccag cagatcgccg cgggcatggt ctacctggcg
tcccagcact 3000tcgtgcaccg cgatttggcc accaggaact gcctggtcgg
ggagaacttg ctggtgaaaa 3060tcggggactt tgggatgtcc cgggacgtgt
acagcactga ctactacagg gtcggtggcc 3120acacaatgct gcccattcgc
tggatgcctc cagagagcat catgtacagg aaattcacga 3180cggaaagcga
cgtctggagc ctgggggtcg tgttgtggga gattttcacc tatggcaaac
3240agccctggta ccagctgtca aacaatgagg tgatagagtg tatcactcag
ggccgagtcc 3300tgcagcgacc ccgcacgtgc ccccaggagg tgtatgagct
gatgctgggg tgctggcagc 3360gagagcccca catgaggaag aacatcaagg
gcatccatac cctccttcag aacttggcca 3420aggcatctcc ggtctacctg
gacattctag gctagggccc ttttccccag accgatcctt 3480cccaacgtac
tcctcagacg ggctgagagg atgaacatct tttaactgcc gctggaggcc
3540accaagctgc tctccttcac tctgacagta ttaacatcaa agactccgag
aagctctcga 3600gggaagcagt gtgtacttct tcatccatag acacagtatt
gacttctttt tggcattatc 3660tctttctctc tttccatctc ccttggttgt
tcctttttct ttttttaaat tttctttttc 3720tttttttttt cgtcttccct
gcttcacgat tcttaccctt tcttttgaat caatctggct 3780tctgcattac
tattaactct gcatagacaa aggccttaac aaacgtaatt tgttatatca
3840gcagacactc cagtttgccc accacaacta acaatgcctt gttgtattcc
tgcctttgat 3900gtggatgaaa aaaagggaaa acaaatattt cacttaaact
ttgtcacttc tgctgtacag 3960atatcgagag tttctatgga ttcacttcta
tttatttatt attattactg ttcttattgt 4020ttttggatgg cttaagcctg
tgtataaaaa agaaaacttg tgttcaatct gtgaagcctt 4080tatctatggg
agattaaaac cagagagaaa gaagatttat tatgaaccgc aatatgggag
4140gaacaaagac aaccactggg atcagctggt gtcagtccct acttaggaaa
tactcagcaa 4200ctgttagctg ggaagaatgt attcggcacc ttcccctgag
gacctttctg aggagtaaaa 4260agactactgg cctctgtgcc atggatgatt
cttttcccat caccagaaat gatagcgtgc 4320agtagagagc aaagatggct
tccgtgagac acaagatggc gcatagtgtg ctcggacaca 4380gttttgtctt
cgtaggttgt gatgatagca ctggtttgtt tctcaagcgc tatccacaga
4440acctttgtca acttcagttg aaaagaggtg gattcatgtc cagagctcat
ttcggggtca 4500ggtgggaaag ccaagaactt ggaaaagata agacaagcta
taaattcgga ggcaagtttc 4560ttttacaatg aacttttcag atctcacttc
cctccgaccc ctaacttcca tgcccacccg 4620tccttttaac tgtgcaagca
aaattgtgca tggtcttcgt cgattaatac cttgtgtgca 4680gacactactg
ctccagacgt cgtttccctg ataggtagag cagatccata aaaaggtatg
4740acttatacaa ttaggggaag ctaatggagt ttattagctg agtatcaatg
tctctgcgtt 4800gtacggtggt gatgggtttt aatgaatatg gaccctgaag
cctggaaatc ctcatccacg 4860tcgaacccac aggactgtgg gaagggcaga
atcaatccct aagggaaagg aaacctcacc 4920ctgagggcat cacatgcact
catgttcagt gtacacaggt caagtccctt gctctgggct 4980ctagttggga
gagtggtttc attccaagtg tactccattg tcagtatgct gtttttgttt
5040ccttcactcc attcaaaaag tcaaaataca aaatttggca cagcatgcca
acgggaggct 5100gtgcccagac caagcactgg aagtgtgctt ctaggcatag
tcattggttt tgcaaaaaga 5160gggctcaaat ttaaatagaa atttacagct
atttgaatgg tcagatatac caagaaagaa 5220aaatatttct gttcctcaag
aaaacttgct accctctgtg aggggaattt tgctaaactt 5280gacatcttta
taacatgagc cagattgaaa gggagtgatt ttcattcatc ttaggtcatg
5340ttatttcata tttgtttctg aaggtgcgat agctctgttt taggttttgc
ttgcgcctgt 5400taattactgg aacaccttat ttttcattaa aggctttgaa
agccaattct caaaaattca 5460aaagtgcaaa ttaacagaac aaaaggaaat
ccagtagcaa ctgcagtcaa gcgagggagt 5520tgacaagata aaccttacgt
ccattcaagt tatatgctgg cctatgagag atgagagttg 5580ggtcgtttgt
tctctttgtt gatgattt 560871930DNAHomo sapiensmodified_base(4)..(4)a,
c, g, t, unknown or other 7tttntttttt tcactgcctg tgccaccagt
tgtgcactgg gccttgcgat cctctcaagc 60tgattcagcc tgcatccttc ccagatggac
acgtgtgtga taaacagctc tgcagtgggg 120tgagggaagg caggggcagc
agggtcctgt atgtcctgcc atctccacaa aagggcagtc 180cttaccccag
ccttgtgctg atgagaccag gcatagacag tcctgacgac acagggcgga
240agggagcagc cattagtgct aatgaggcag gcggcctgaa agctttgtac
tctgcagtgg 300ctcgcccacc cagggaacag ttcgttctgt ttccttggct
tccaggaacc ctaggcagaa 360aggggtttgg ggacaggagc aggagtgggc
ggtcttggag aaacctggag ggagaaaggg 420aggggaggac cagaaatgta
gtcaggaggg cctaggattg gttaggtggg cttttccttc 480ccctttccct
ccaaagaaac ccaggttctg gttctgcacc tacccctgcc caacagtggc
540cattggccca tcacccgctc caatgtcctt gacccgaatt cttggaagca
caggaaacaa 600catgccacat aggggttgag taagcatctc tggggccaca
aattaaatta agctttcagg 660gccgcctgcc ttgttattgc taatggttct
agccctgctc agctcctagg tccctgtcct 720gtggaaattt gtggaccctg
ggcaccctct cttgctccca aattttaatc ggctcctgga 780aacctcaccc
caaattggag ataggcactc ctcttgtaga acaaaaggct caggttcagg
840gagtgagggc ctgaactgtg cccccaccct ccaggaaggg tccttcacgg
cctggctgca 900gggatcagtc acgtgtggcc cttcattagg ccctgccata
taagccaagg gcacggggtg 960gccgggaact ctctaggcaa gaatcccgga
ggcagaggtg agtcctcagg ttgggcaggg 1020actcctcctc tctgtggggt
ctctatctgg gcacctagag gggactccaa ggataaggag 1080ggactaagtg
gtacatcttc ctgctgagcc aggccatgct gaccgcagcg gtgctgagct
1140gtgccctgct gctggcactg cctgccacgc gaggagccca gatgggcttg
gcccccatgg 1200agggcatcag aaggcctgac caggccctgc tcccagagct
cccaggtcag tgtgagcaag 1260ggtgggactg ggcggggcct gaataccctc
tggccacaaa tagtctcccc tggcataaac 1320cctctttctc ccttcccaaa
ccctcccctg ggaggtgggt gctttgtgca tgggggttcc 1380tgccctcaca
tcctctgccc caggcctggg cctgcgggcc ccactgaaga agacaactgc
1440agaacaggca gaagaggatc tgttgcagga ggctcaggcc ttggcagagg
taactgctca 1500gggaaaaggg taaggtggtg gcccttggga gggggcattg
ggtattagct cctctcccca 1560gctccaaact ccctcaccag cgacgacact
accgaccacc ccttcccatg ctccactgcc 1620atcctgcaca ggttgggaca
ggtaagatcc ctggatctgt ctttagaggc ctgtgctggt 1680tccccacccc
tgcaggtact agacctgcag gaccgcgagc cccgctcctc acgtcgctgc
1740gtaaggctgc atgagtcctg cctgggacag caggtgcctt gctgtgaccc
atgtgccacg 1800tgctactgcc gcttcttcaa tgccttctgc tactgccgca
agctgggtac tgccatgaat 1860ccctgcagcc gcacctagct ggccaacgtc
agggtcgggg caaggaaact cgaataaagg 1920atgggaccaa
19308594DNAUnknownDescription of Unknown Woodchuck post-
transcriptional regulatory element polynucleotide 8ataatcaacc
tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 60ctccttttac
gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
120gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
tatgaggagt 180tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
gtttgctgac gcaaccccca 240ctggttgggg cattgccacc acctgtcagc
tcctttccgg gactttcgct ttccccctcc 300ctattgccac ggcggaactc
atcgccgcct gccttgcccg ctgctggaca ggggctcggc 360tgttgggcac
tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
420tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
ccttcggccc 480tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
tctgcggcct cttccgcgtc 540ttcgccttcg ccctcagacg agtcggatct
ccctttgggc cgcctccccg catc 594921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 9aatactgtca
cacacgctca g 2110247PRTHomo sapiens 10Met Thr Ile Leu Phe Leu Thr
Met Val Ile Ser Tyr Phe Gly Cys Met1 5 10 15Lys Ala Ala Pro Met Lys
Glu Ala Asn Ile Arg Gly Gln Gly Gly Leu 20 25 30Ala Tyr Pro Gly Val
Arg Thr His Gly Thr Leu Glu Ser Val Asn Gly 35 40 45Pro Lys Ala Gly
Ser Arg Gly Leu Thr Ser Leu Ala Asp Thr Phe Glu 50 55 60His Val Ile
Glu Glu Leu Leu Asp Glu Asp Gln Lys Val Arg Pro Asn65 70 75 80Glu
Glu Asn Asn Lys Asp Ala Asp Leu Tyr Thr Ser Arg Val Met Leu 85 90
95Ser Ser Gln Val Pro Leu Glu Pro Pro Leu Leu Phe Leu Leu Glu Glu
100 105 110Tyr Lys Asn Tyr Leu Asp Ala Ala Asn Met Ser Met Arg Val
Arg Arg 115 120 125His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val
Cys Asp Ser Ile 130 135 140Ser Glu Trp Val Thr Ala Ala Asp Lys Lys
Thr Ala Val Asp Met Ser145 150 155 160Gly Gly Thr Val Thr Val Leu
Glu Lys Val Pro Val Ser Lys Gly Gln 165 170 175Leu Lys Gln Tyr Phe
Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr 180 185 190Lys Glu Gly
Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln Cys 195 200 205Arg
Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys 210 215
220Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys
Thr225 230 235 240Leu Thr Ile Lys Arg Gly Arg 24511838PRTHomo
sapiens 11Met Ser Ser Trp Ile Arg Trp His Gly Pro Ala Met Ala Arg
Leu Trp1 5 10 15Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala
Phe Ala Cys 20 25 30Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp
Cys Ser Asp Pro 35 40 45Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu
Pro Asn Ser Val Asp 50 55 60Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala
Asn Gln Lys Arg Leu Glu65 70 75 80Ile Ile Asn Glu Asp Asp Val Glu
Ala Tyr Val Gly Leu Arg Asn Leu 85 90 95Thr Ile Val Asp Ser Gly Leu
Lys Phe Val Ala His Lys Ala Phe Leu 100 105 110Lys Asn Ser Asn Leu
Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr 115 120 125Ser Leu Ser
Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile 130 135 140Leu
Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys145 150
155 160Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr
Cys 165 170 175Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu
Gln Ile Pro 180 185 190Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala
Pro Asn Leu Thr Val 195 200 205Glu Glu Gly Lys Ser Ile Thr Leu Ser
Cys Ser Val Ala Gly Asp Pro 210 215 220Val Pro Asn Met Tyr Trp Asp
Val Gly Asn Leu Val Ser Lys His Met225 230 235 240Asn Glu Thr Ser
His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser 245 250 255Ser Asp
Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val 260 265
270Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr
275 280 285Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys
Ile Pro 290 295 300Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln
Trp Phe Tyr Asn305 310 315 320Gly Ala Ile Leu Asn Glu Ser Lys Tyr
Ile Cys Thr Lys Ile His Val 325 330 335Thr Asn His Thr Glu Tyr His
Gly Cys Leu Gln Leu Asp Asn Pro Thr 340 345 350His Met Asn Asn Gly
Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly 355 360 365Lys Asp Glu
Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile 370 375 380Asp
Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr385 390
395 400Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn
Glu 405 410 415Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu
His Leu Ser 420 425 430Val Tyr Ala Val Val Val Ile Ala Ser Val Val
Gly Phe Cys Leu Leu 435 440 445Val Met Leu Phe Leu Leu Lys Leu Ala
Arg His Ser Lys Phe Gly Met 450 455 460Lys Asp Phe Ser Trp Phe Gly
Phe Gly Lys Val Lys Ser Arg Gln Gly465 470 475 480Val Gly Pro Ala
Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro 485 490 495Leu His
His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly 500 505
510Gly Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu
515 520 525Asn Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro
Asp Thr 530 535 540Phe Val Gln His Ile Lys Arg His Asn Ile Val Leu
Lys Arg Glu Leu545 550 555 560Gly Glu Gly Ala Phe Gly Lys Val Phe
Leu Ala Glu Cys Tyr Asn Leu 565 570 575Cys Pro Glu Gln Asp Lys Ile
Leu Val Ala Val Lys Thr Leu Lys Asp 580 585 590Ala Ser Asp Asn Ala
Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu 595 600 605Thr Asn Leu
Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val 610 615 620Glu
Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp625 630
635 640Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met
Ala 645 650 655Glu Gly Asn Pro Pro Thr Glu Leu Thr Gln Ser Gln Met
Leu His Ile 660 665 670Ala Gln Gln Ile Ala Ala Gly Met Val Tyr Leu
Ala Ser Gln His Phe 675 680 685Val His Arg Asp Leu Ala Thr Arg Asn
Cys Leu Val Gly Glu Asn Leu 690 695 700Leu Val Lys Ile Gly Asp Phe
Gly Met Ser Arg Asp Val Tyr Ser Thr705 710 715 720Asp Tyr Tyr Arg
Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met 725 730 735Pro Pro
Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val 740 745
750Trp Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln
755 760 765Pro Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile
Thr Gln 770 775 780Gly Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln
Glu Val Tyr Glu785 790 795 800Leu Met Leu Gly Cys Trp Gln Arg Glu
Pro His Met Arg Lys Asn Ile 805 810 815Lys Gly Ile His Thr Leu Leu
Gln Asn Leu Ala Lys Ala Ser Pro Val 820 825 830Tyr Leu Asp Ile Leu
Gly 83512132PRTHomo sapiens 12Met Leu Thr Ala Ala Val Leu Ser Cys
Ala Leu Leu Leu Ala Leu Pro1 5 10 15Ala Thr Arg Gly Ala Gln Met Gly
Leu Ala Pro Met Glu Gly Ile Arg 20 25 30Arg Pro Asp Gln Ala Leu Leu
Pro Glu Leu Pro Gly Leu Gly Leu Arg 35 40 45Ala Pro Leu Lys Lys Thr
Thr Ala Glu Gln Ala Glu Glu Asp Leu Leu 50 55 60Gln Glu Ala Gln Ala
Leu Ala Glu Val Leu Asp Leu Gln Asp Arg Glu65 70 75 80Pro Arg Ser
Ser Arg Arg Cys Val Arg Leu His Glu Ser Cys Leu Gly 85 90 95Gln Gln
Val Pro Cys Cys Asp Pro Cys Ala Thr Cys Tyr Cys Arg Phe 100 105
110Phe Asn Ala Phe Cys Tyr Cys Arg Lys Leu Gly Thr Ala Met Asn Pro
115 120 125Cys Ser Arg Thr 130
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